ABSTRACT OF DISSERTATION
Sameera Dasari
The Graduate School
University of Kentucky
2007
INFLUENCE OF THE SEROTONERGIC SYSTEM ON PHYSIOLOGY,
DEVELOPMENT, AND BEHAVIOR OF DROSOPHILA MELANOGASTER
__________________________________________
ABSTRACT OF DISSERTATION
_____________________________________
A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the
College or Arts and Sciences at the University of Kentucky
By
Sameera Dasari
Lexington, Kentucky
Director: Dr. Robin Lewis Cooper, Associate Professor of Biology
Lexington, Kentucky
2007
Copyright © Sameera Dasari 2007
ABSTRACT OF DISSERTATION
INFLUENCE OF THE SEROTONERGIC SYSTEM ON PHYSIOLOGY,
DEVELOPMENT, AND BEHAVIOR OF DROSOPHILA MELANOGASTER
The regulation and modulation of the serotonergic system is clinically significant in humans. Abnormally low levels of serotonin can result in depression and conditions like panic disorder, obsessive-compulsive disorder, social anxiety disorder, sudden infant death syndrome, and eating disorders. The mechanistic role of serotonin (5-HT) on the neural circuits related with these diseases is not definitively known.
Drosophila is a simple model system that provides an advantage over vertebrates to modify genetically and for electrophysiological studies on identifiable cells. In this organism the sensory-CNS-motor circuit is modulated by 5-HT, octopamine (OA), and dopamine (DA), which gives one insight that these neuromodulators are playing a role in central neuronal circuits. The role of 5-HT in the behavior and development of Drosophila melanogaster larvae is being studied. p-CPA (para-chlorophenylalanine) blocks the synthesis of 5-HT by inhibiting tryptophan hydroxylase. The development, behavior and physiology in 3rd instar larvae are affected after feeding this drug. MDMA (3,4 methylenedioxyamphetamine), an analog of methamphetamine is a drug of abuse that has been shown to cause depletion of 5-HT from nerve terminals. It causes the 5-HT transporter to work in reverse. Thus, a dumping of 5-HT results. In Drosophila 3rd instar larva development, physiology and behavior are effected when MDMA is fed throughout their development period. Also at the fly neuromuscular junction, (NMJ) MDMA is causing more evoked vesicular release of glutamate from the presynaptic nerve terminal. Also using anti-sense expression of the 5-HT2dro receptor, role of 5-HT and one of its receptors is studied on development, physiology and behavior. Knock down of 5-HT2dro resulted in developmental delay. Physiology and behavior were also abnormal in these animals.
KEYWORDS: Serotonin, Drosophila, sensory-CNS-circuit, MDMA, heart rate.
Sameera Dasari January 19, 2007
iv
INFLUENCE OF THE SEROTONERGIC SYSTEM ON PHYSIOLOGY,
DEVELOPMENT, AND BEHAVIOR OF DROSOPHILA MELANOGASTER
By
Sameera Dasari
Dr. Robin L. Cooper Director or Dissertation
Dr. Brian C. Rymond
Director of Graduate Studies
January 19, 2007
RULES FOR THE USE OF DISSERTATIONS
Unpublished dissertations submitted for the Doctor's degree and deposited in the University of Kentucky Library are as a rule open for inspection, butare to be used only with due regard to the rights of the authors. Bibliographical references may be noted, but quotations or summaries of parts may be published only with the permission of the author, and with the usual scholarly acknowledgments. Extensive copying or publication of the dissertation in whole or in part also requires the consent of the Dean of the Graduate School of the University of Kentucky. A library that borrows this dissertation for use by its patrons is expected to secure the signature of each user.
DISSERTATION
Sameera Dasari
The Graduate School
University of Kentucky
2007
INFLUENCE OF THE SEROTONERGIC SYSTEM ON PHYSIOLOGY, DEVELOPMENT, AND BEHAVIOR OF DROSOPHILA MELANOGASTER
__________________________________________
DISSERTATION __________________________________________
A dissertation submitted in partial fulfillment of the
requirements for the degree of Doctor of Philosophy in the College of Arts and Sciences at the
University of Kentucky
By Sameera Dasari
Lexington, Kentucky
Director: Dr. Robin Lewis Cooper, Associate Professor
Lexington, Kentucky
2007
Copyright © Sameera Dasari 2007
iii
ACKNOWLEDGEMENTS
I acknowledge all those that helped in making this dissertation possible.
First and foremost I thank my advisor, Robin L. Cooper for his guidance and
patience for the last 4 and half years. Knowledge and skills I acquired in this field
during this peiord and putting together this dissertation would not have been
possible without his help. Also thanks to my committee members, Drs. Douglas
Harrison, John Rawls and Sidney Whiteheart for their help and time to complete this
dissertation.
I thank my parents and sister for their love and encouragement through this
process and throughout my carrer. I thank my husband, Raju for his love and
support and making sure I stay on track and sane. I thank my family for their love
and support through this process.
I also, must acknowledge and thank my graduate lab mates, Andrew
Johnstone and Mohati Desai, who were always available for advice and/or moral
support for which I am indebted to. Thanks also to the numerous undergraduates in
the lab who were a constant source of entertainment for the last four years. Special
thanks to a high school student then A. Clay Turner, and undergraduate Blaire
Culman-Clark for assisting in projects that are part of this dissertation.
Finally thanks to all the graduate students and friends in the biology program.
Especially, Karthik Venkatachalam, Sakshi Pandit and Scott Frasure, who without
their moral support, my sanity may not have been possible.
iv
TABLE OF CONTENTS
ACKNOWLEDGEMENTS ......................................................................................... III
TABLE OF CONTENTS............................................................................................ IV
LIST OF TABLES.....................................................................................................VII
LIST OF FIGURES .................................................................................................VIII
LIST OF FILES ..........................................................................................................X
CHAPTER 1............................................................................................................... 1
INTRODUCTION ....................................................................................................... 1 GENERAL BACKGROUND OF DEVELOPMENT ..................................................... 1
NEURAL ACTIVITY IN DEVELOPMENT................................................................... 4
INFLUENCE OF HORMONES AND NEUROMODULATORS ON DEVELOPMENT. 6
SEROTONIN (5, HYDROXY TRYPTAMINE, 5-HT).................................................. 7
ROLE OF 5-HT IN DEVELOPMENT.......................................................................... 9
EFFECTS ON DEVELOPMENT THROUGH RECEPTORS.................................... 12
MANIPULATIONS IN NEUROMODULATOR SYSTEMS ON FUNCTION .............. 13
5-HT RECEPTORS AND EXPRESSION IN DROSOPHILA.................................... 17
USING THE DROSOPHILA HEART AS A BIOASSAY FOR 5-HT EFFECTS......... 19
CHAPTER 2............................................................................................................. 23
MODULATION OF SENSORY-CNS-MOTOR CIRCUITS BY SEROTONIN, OCTOPAMINE, AND DOPAMINE IN SEMI-INTACT DROSOPHILA LARVA.......... 23 ABSTRACT.............................................................................................................. 23
INTRODUCTION ..................................................................................................... 24
METHODS............................................................................................................... 26
RESULTS ................................................................................................................ 27
DISCUSSION........................................................................................................... 31
CHAPTER 3............................................................................................................. 42
INFLUENCE OF P-CPA AND MDMA ON THE SEROTONERGIC SYSTEM IN RELATION TO PHYSIOLOGY, DEVELOPMENT AND BEHAVIOR OF DROSOPHILA MELANOGASTER........................................................................... 42
v
ABSTRACT.............................................................................................................. 42
INTRODUCTION ..................................................................................................... 42
METHODS............................................................................................................... 44
RESULTS ................................................................................................................ 48
DISCUSSION........................................................................................................... 54
CHAPTER 4............................................................................................................. 71
KNOCK DOWN OF 5-HT2 RECEPTORS ALTERS DEVELOPMENT, BEHAVIOR AND CNS ACTIVITY IN DROSOPHILA MELANOGASTER.................................... 71 ABSTRACT.............................................................................................................. 71
INTRODUCTION ..................................................................................................... 71
METHODS............................................................................................................... 74
RESULTS ................................................................................................................ 77
DISCUSSION........................................................................................................... 83
CHAPTER 5........................................................................................................... 100
DIRECT INFLUENCE OF SEROTONIN ON THE LARVAL HEART OF DROSOPHILA MELANOGASTER......................................................................... 100 ABSTRACT............................................................................................................ 100
INTRODUCTION ................................................................................................... 101
METHODS............................................................................................................. 104
RESULTS .............................................................................................................. 108
DISCUSSION......................................................................................................... 112
CHAPTER 6........................................................................................................... 123
DISCUSSION......................................................................................................... 123
REFERENCES ...................................................................................................... 136
CHAPTER 1........................................................................................................... 136
CHAPTER 2........................................................................................................... 161
CHAPTER 3........................................................................................................... 169
CHAPTER 4........................................................................................................... 182
CHAPTER 5........................................................................................................... 191
CHAPTER 6........................................................................................................... 202
vi
VITA....................................................................................................................... 214
vii
LIST OF TABLES
TABLE 5.1……………………………………………………………………………123
viii
LIST OF FIGURES
Figure 2.1: Schematic diagram of the Drosophila larva preparation: ....................... 35
Figure 2.2: Representative traces of induced responses recorded in muscle 6 in
various segments..................................................................................................... 36
Figure 2.3: Recuritment of motor units..................................................................... 38
Figure 2.4: The influence of neuromodulators in altering the sensory to motor neuron
central circuit was examined.................................................................................... 40
Figure 3.1: p-PCA growth curve............................................................................... 63
Figure 3.2: MDMA growth Curve.............................................................................. 64
Figure 3.3: Body wall and mouth hook contractions................................................. 65
Figure 3.4: Spontaneous activity in 3rd instar CS larvae. ....................................... 66
Figure 3.5: Spontaneous activity.............................................................................. 67
Figure 3.6: Sensory-CNS-motor circuit. ................................................................... 68
Figure 3.7: Heart rate............................................................................................... 69
Figure 3.8: HPLC analysis of 3rd instar larvae......................................................... 70
Figure 4.1: Locomotory movements at room temperature. ...................................... 90
Figure 4.2: Locomotory movements of larvae that are grown at high temperature. . 91
Figure 4.3: Locomotory movements at high temperature......................................... 92
Figure 4.4: Locomotory movements at 31-32C........................................................ 93
Figure 4.5: Development curve for low temperature. ............................................... 94
Figure 4.6: Development curve for room temperature. ............................................ 95
Figure 4.7: Development curve for high temperature............................................... 96
Figure 4.8: Spontaneous activity.............................................................................. 97
Figure 4.9: Sensory-CNS-motor circuit .................................................................... 98
ix
Figure 4.10: Heart rate............................................................................................. 99
Figure 5.1: Dorsal Vessel....................................................................................... 120
Figure 5.2: Heart rate of 3rd instar semi-intact preparations.................................. 121
Figure 5.3: The effects of 5-HT on intact and on the isolated heart and aorta
segments. .............................................................................................................. 122
x
LIST OF FILES Dasari.pdf 4.5mb
xi
1
CHAPTER 1
INTRODUCTION
GENERAL BACKGROUND OF DEVELOPMENT Developmental neurobiology is the study of how neurons grow and form
connections. This includes understanding development and complex organization of
the brain and its systems as well as factors affecting this process. This involves
evaluating the sensory inputs and processing of those signals that are then
responsible for defined motor patterns or behaviors.
The CNS during development is known to be dependent on the on-going
electrical activity and synaptic transmission. Thus, the activity of sensory input to
drive interneurons, which in return drives motor neurons and their targets are critical.
Not only is activity important for developing a sensory-CNS-motor circuit but within
the CNS the various internal circuits also require activity to establish themselves.
The nervous system (NS) is very dynamic during development and depending on
the animal model there can be a wide range in the rate of development of the NS.
The CNS develops at different rates during the early life stages among animal
species. In addition, particular regions of the NS develop at various rates. As one
might expect vegetative functions such as regulation of the heart, blood pressure
and breathing to develop earlier as compared to those for fine motor coordination,
visual or olfactory senses.
Recently there is a vibrant interest in understanding more about neuronal
replacement and treatments with stem cells that differentiate into neurons within the
adult mammalian CNS (Encinas et al., 2006; Huang and Herbert, 2006). It is now
2
established that in the adult mammalian CNS there are cells that can migrate into
neural tissue and start to grow processes, which connect into existing circuits (Lie et
al., 2004; Hagg, 2005; Miller, 2006; Gage 1995; McDonald, 1999). It was shown in
1983 (Goldman and Nottebohm) that many new neurons are formed in adult song
bird (canary) brain and that seasonal changes occur in singing birds resulting in new
neurons that arise from stem cells, which became part of the neural circuitry involved
with vocalization (Nottebohm et al., 1986). What is truly amazing is that development
regresses over the winter and repeats itself the following year (Nottebohm et al.,
1986). Along with research that is ongoing in birds, other groups (Monfils et al.,
2006) discovered that when cells in the CNS are damaged in rodents parts of it filled
back in with new neurons. It is now known that the sub-ventricular zones contain
stem cells that could indeed transform into neurons and help repair damaged neural
tissue (Lois and Alvarez-Buylla, 1993; Luskin, 1993). This brings one to the point of
maintenance of the existing neural circuits by replacement after the initial
establishment of the CNS. At the NMJ in adult rodents it has been known for
sometime that synapses are not hard wired but are very dynamic in pairing back and
re-growing at normal NMJs that are not undergoing regeneration or repair (see
review Sanes and Lichtman, 1999; Purves and Lichtman, 1987).
Expanding on these earlier findings I wished to address if one altered
synaptic communications by neuromodulators in the larval brain of Drosophila would
there be consequences in further development or maintenance of functional neural
circuits. As compared to other animal models Drosophila offers a rapidly developing
CNS and this animal model serves as a spring board for genetic studies to address
3
similar questions that are being addressed in vertebrates by targeting specific genes
and proteins.
In order to relay key aspects in neural development I will give a brief
overview next on the mammalian systems since a wealth of information is available
and some of the underlying principles are important for all animals. Mammalian
neurogenesis begins with the formation of the neural plate that is a thickening of
ectodermal cells on the dorsal aspect of the developing embryo. Ridges are formed
at the lateral edges of the plate, which curl up to meet at the dorsal midline to form
the neural tube. The internal cavity created by the tube is called the ventricle. As
closure of the neural tube is occurring, specialized regions of the nervous system
begin to emerge through differential cell division and migration. Major subdivisions of
brain include the mylencephalon and metencephalon, the mesencephalon, and the
prosencephalon, which matures into the diencephalon and telencephalon. Through
this process, the subdivision of the developing brain lays the foundation for regional
specialization in the mature brain. By the end of embryonic stages of an animal,
neurons make connections with other neurons either locally or at distant central or
peripheral target tissues. For example, retinal ganglion axons from the eyes enter
the brain at the junction of optic nerve and diverge to the optic tectum and lateral
geniculate nucleus. The numerous synapses and connections that are made go
through the process of refinement, rearrangement and elimination are based on
activity (Wiesel and Hubel , 1965; Levay et al., 1980). As for the vertebrate brain, the
Drosophila larval brain also shows regions of similar function that can be quantified
and examined for alterations in size (Iyengar, et al., 2006). Possible in the near
4
future synaptic connectivity within defined circuits will also be addressed.
NEURAL ACTIVITY IN DEVELOPMENT
Development and maintenance of neural circuits is dependent on the
electrical activity. There are 2 general activities in the brain that can effect the
development of the neural circuits – spontaneous activity that is devoid of any
sensory or motor input and activity based on experience that is from input of sensory
and motor units. Spontaneous activity is seen as bursts of activity for a few seconds
or minutes in absence of neuronal stimulation. This activity was shown to have an
effect on both synapse formation and elimination. For example, when newborn cats
were deprived of any visual activity by closing both eyes; ocular dominance columns
for both the eyes are still formed although the columns are obscured (Hubel and
Wiesel 1965; Sherman and Spear, 1982). This was thought to be due to
spontaneous activity. To prove this TTX (a blocker for sodium channels) was
injected into both eyes of 2-6 weeks postnatal kittens. The experiment demonstrated
that the lateral geniculate nucleus did not segregate into stripes (Stryker and Harris,
1986). This kind of activity was also seen in the developing auditory system of birds
and the spinal cord of chicks (Lippe, 1994; O’Donovan et al., 1994; Kotak and
Sanes, 1995).
Experienced based activity or use-dependent activity involvement in the
development of neural circuits was shown by the pioneering work of Hubel and
Wiesel (1963a,b) for which they received a Nobel Prize. Their work on visual
deprivation in one eye of newborn cats showed that cortical neurons did not
5
responded to stimulation from the closed eye. In normal animals half the cortical
neurons respond to one eye and the other half to the other eye. Also the closure of
lids for 3 months led to blindness in the newborn cats and monkeys (Wiesel and
Hubel, 1963b; Wiesel 1982). At the same time Hubel and Wiesel showed that the
cortical region of the CNS, which would have been supplied by the deprived eye,
shrunk whereas those of the other eye expanded. These results have shown cortical
neurons in the visual system are developed and maintained based on activity. Since
then many studies have shown that spontaneous electrical activity during the
embryonic stages and experience based activity in early postnatal stages are
important for the development and refining of the neural circuits (Penn and Shaatz
1999; Zhang and Poo� 2001).
However similar experiments carried out in adult animals had no effect on
their ocular columns architecture or even the responses from cortical neurons and
on blindness (Wiesel, 1982; LeVay et al., 1980). Hence they concluded that the age
of animal when these experiments were conducted were important. The plasticity in
the newborn animals is lost as the animal ages. The time at which the plasticity can
occur is referred to as the "critical period". The critical period is defined as, period in
early stages of development of an animal where it shows very high sensitivity to the
external stimuli and experience. Critical periods are seen in many animals and in
many sensory systems such as visual, auditory, sound localization, bird song, and
olfaction. Critical periods are altered by various chemical compounds like hormones,
neurotransmitters or neuromodulators and drugs of abuse like cocaine.
6
INFLUENCE OF HORMONES AND NEUROMODULATORS ON DEVELOPMENT
Hormones and neuromodulators are chemical compounds, which have many
roles in an organism and are known to affect and regulate development of the whole
animal as well as the nervous system. Hormones are known to regulate fate of some
neurons in certain areas of the CNS, as well demonstrated in songbirds. The higher
vocal center is more developed in males than females and thus the effects of
testosterone were investigated for its role on growth of this key neural location. This
center plays an important role in song acquisition and retention. It was shown that
when female birds are injected with testosterone, the female could be induced to
sing like a male (Nottebohm and Arnold, 1976; Nottebohm, 1980). In insects, it is
well established that hormones such as ecdysone and juvenile hormone alter neural
development and differentiation (Garen et al, 1977; Pak and Gilbert, 1987; Truman,
1996). The surge of ecdysone in the pupal stage of Drosophila likely plays a key role
in inducing gross alterations in the neural circuitry (Kraft et al, 1998; Thummel, 1996;
Truman and Reiss, 1988) and motor unit function (Li and Cooper, 2001; Li et al,
2001). It has also been demonstrated that the sequence of exposure of
neuromodulators (serotonin and octopamine) and cocktails produce differential
effects on synaptic modulation in other arthropods (i.e., the crustaceans) (Djokaj et
al, 2001). Cocktails of various hormones and neuromodulators have not yet been
investigated for their combined effects in developmental roles.
It is shown in various studies that neurotransmitter signaling is present before
synaptogenesis (reviewed in Herlenius and Lagercrantz, 2004). But total knock-out
of synaptic trafficking in mouse was shown to have no effect on the formation of
7
brain structure or synapses, however for survival of these synapses synaptic activity
is needed (Verhage et al., 2000). Different neurotransmitters have different roles in
the process of the brain development. For example, the noradrenergic system (nor-
epinephrin) is essential for the brain development as it regulates the development of
Cajal-Retzius cells, which are the first neurons to be formed. Cajal-Retzius cells are
important for the migration of neuronal cells and laminar formation (Naqui et al.,
1999,). Also a surge of norepinephrin at birth is important for formation of olfactory
system and learning, that is important for recognition of one’s mother (Insel and
Young, 2001). Similarly 5-HT has been shown to affect neuronal differentiation,
migration and synaptogenesis (Gaspar et al., 2003), acetylcholine (Ach) mediates
synaptic connections and wiring of the circuits (Maggi et al., 2003), dopamine (DA)
neurons appear in gestational period of development in rats (Olson and Seiger,
1972; Herlenius and Lagercrantz, 2004), humans (Sundstrom et al., 1993). Any
disturbance in the development of dopaminergic system leads to various diseases
like dyskinesia, obsessive compulsive disorder, etc (Zhou et al., 1995a,b). Other
neuromodulators, which also serve as neurotransmitters, have also been of interest.
Octopamine (OA), which is not found in vertebrates but is in invertebrates, has gain
much attention because of its dramatic effect on behavior and development,
particularly in insects (Barron et al., 2002; Schulz et al., 2002; Fox et al., 2006;
reviewed in Roeder, 1999; Osborne, 1996; Monastirioti, 1999).
SEROTONIN (5, HYDROXY TRYPTAMINE, 5-HT)
One of the main neurotransmitters and neuromodulator that has been
targeted over the years is 5-HT. It was identified as early as in 1930’s with the name
8
enteramine and later name serotonin (Whitaker-Azmitia, 1999). The compound is
commonly found in very simple to complex invertebrates and has been suggested to
even be one of the 1st neurotransmitters in the evolution of animals (Whitaker-
Azmitia, 1999). The role of 5-HT in invertebrates has been investigated for some
time and is known to alter sensory, CNS and motor function (e.g., Marinesco and
Carew, 2002) and it may even serve as an overall enhancer for animals similar to
epinephrine via the sympathetic nervous system in mammals (review- Shuranova et
al., 2006). Recently 5-HT is shown to have a role in mediating the structure of brain
and also in neurogenesis (Yan et al 1997, Gould, 1999). Also for human and
mammalian studies the role of 5-HT and its various receptor subtypes gained
interest when it was established that medicinal herbs and synthesized compounds,
like LSD, targeted 5-HT receptors which were responsible for altered behaviors
(Nichols, 2004; Reissig et al., 2005; Gresch et al., 2005).
Rapport et al., (1948b) were the first investigators to show the structure of 5-
HT and establish it as a vasoconstrictor (Rapport et al., 1948a). Twarog and Page
(1953) showed for the first time that 5-HT is present in brain using dog, rat and rabbit
brain extracts. Woolley and Shaw (1954) suggested 5-HT to have a role in brain
development, as it is similar to the auxins, a plant growth hormone. Later Gaddum
and Picarelli (1957) started reporting on various 5-HT receptors. To date seven
classes of 5-HT receptors are known and classified pharmacologically into 14
distinct subtypes of mammalian receptors (Barnes and Sharp, 1999, Hoyer et al.,
2002). 13 of these receptors belong to the category of G-protein coupled receptors
(GPCR) and one receptor (5-HT3) is a ligand-gated ion channel type of receptor.
9
In considering comparative studies based on pharmacology and genomic
sequence data there are a considerable number of 5-HT receptor subtypes (Barnes
and Sharp; 1999, Hoyer et al., 2002) and some share similar sequence homology
between species (Barnes and Sharp, 1999; Saudou and Hen, 1994). Thus, using
one model organism in examining regulation of particular receptor subtypes may
shed light into the functions of other organisms, such as humans where such
experimentation is problematic to investigate developmental topics.
ROLE OF 5-HT IN DEVELOPMENT
In general synaptic plasticity is the ability of synapses between two neurons
or a neuron to a target cell to change in strength or number of connections in the
network. There are various mechanisms by which synaptic plasticity is measured,
such as how much neurotransmitter is released at the synapse or the
responsiveness on the receiving cell. Even structural changes that occur in the
circuit would be a form of synaptic plasticity. The underlying cause of behavioral
plasticity is assumed to be due to synaptic plasticity within the neural circuitry of an
animal. Gaspar et al (2003) has shown that 5-HT uptake is necessary for the normal
development and refining of cortical sensory maps during the critical period of
development in mouse. Role of 5-HT in synaptic plasticity has been shown in
rodents (Mnie-Filali et al., 2006), chickens (Chen et al., 1997), Aplysia (Marinesco et
al., 2004; Chang et al., 2003), and crustaceans (Harzsch et al., 1999; Cooper et al.,
2003). Not only are there direct effects on electrical activity by 5-HT on neurons but
indirect effects on the whole system. For example, it is known that 5-HT can alter the
release of growth hormone in rats (Murakami et al., 1986) which then alters the
10
growth of the entire animal. In humans the effect of 5-HT on growth hormone is not
well known. Studies have shown stimulatory (Mota et al., 1995), inhibitory
(Casanueva et al., 1984) and no effect (Handdwerger et al., 1975) of 5-HT on
secretion of GH. Such global effects are also known to occur even invertebrates
such as crustaceans in which 5-HT can have an effect on the release of the
hyperglycemic hormone (Lee et al., 2000, 2001; Escamilla-Chimal et al., 2002).
The levels of 5-HT during the development are very important. Either high or
low levels of 5-HT during the critical period can lead to miswiring of connections
(Gaspar et al., 2003). Miswiring of neurons can lead to various problems like drug
addiction disorders, anxiety disorders and autism. The levels of 5-HT during the fetal
stages and in young children are high and the levels come down as development
progresses. But in autistic children the levels of 5-HT is maintained high (Chugani,
2002; Warren and Singh, 1996; Hanley et al., 1977). 5-HT is also associated with
other disorders like anti-social behavior, depression, migraine etc. The drugs of
abuse like cocaine, MDMA (3,4-methylenedioxymethamphetamine, ecstasy), LSD or
even anti-depressants like SSRI when taken by pregnant women can effect the
development and behavior of the offspring (Discussed in Chapter 1, section vii and
chapter 3).
To illustrate the role of 5-HT, the serotonin-ergic system is commonly
manipulated using pharmacological agents. A few pharmacological appraoches
used to deplete 5-HT in vivo are to block tryptophan from being used to make 5-HT
(Drummond, 2006; Hood et al., 2006). Another method is to kill the seretonergic
neurons using the neurotoxin 5,7-dihydroxytryptamine (5,7 DHT; Walker et al., 2006;
11
Shirahata et al., 2006; Jha et al., 2006). The use of p-chloroamphetamine (PCA;
Eide et al., 1988) and p- chlorophenylalanine (PCPA), which are both inhibitors of
rate limiting enzyme tryptophan hydroxylase in the 5-HT biosynthesis pathway (Jha
et al., 2006; Cooper et al., 2001) are assumed to be a specific inhibitor of 5-HT
biosynthesis. However, recent studies have shown PCPA to casue a significant
effect on another neurotransmitter, norepinehrine (Jha et al., 2006; Dailly et al.,
2006). Also the initial study that showed PCPA as depletor of 5-HT has reported
small levels of dopamine and norepinephrine to be reduced in brain tissue (Koe and
Weissman, 1966; Sanders-Bush and Massari 1977). The levels of 5-HT are
increased by 5-hydroxytryptophan (5-HTP), immediate precursor of 5-HT in
biosynthesis pathway (Pellegrino and Bayer, 2000; Fickbohm et al., 2005).
PCPA has been successfully used as a 5-HT depletor for many years and in
many organisms like rats (Sinha, 2006; Jha et al., 2006; Koe and Wiessman, 1966),
mouse (Khozhai and Otellin, 2006; Dailly et al., 2006; Koe and Wiessman, 1966),
dog (Haga et al., 1996; Dourish et al., 1986; Koe and Wiessman 1966), Drosophila
(Banerjee at al., 2004; Pendelton et al., 2002; Vaysse et al., 1988; Kamyshev et al.,
1983), snail (Filla et al., 2004; Baker and Croll 1996; Baker et al., 1993), and
crustaceans (Mattson and Spaziani, 1986; Cooper et al., 2001).
In Drosophila, PCPA was first used to study the role of 5-HT on locomotion of
Canton-S (CS) adults by Kamyshev et al. (1983). They showed that locomotor
acitivity increases upon administration of PCPA at 150 µg/ml of yeast-raisin media.
Vaysse et al (1988) used 0.6g/ L of PCPA to study the learning behavior in
12
Drosophila. In Chapter 3 I report in detail the effects of PCPA on development,
locomotor behavior and physiology of larval Drosophila system.
EFFECTS ON DEVELOPMENT THROUGH RECEPTORS
Generally hormones/neuromodulators bring about their biological responses
by interacting with their receptors. Many different studies have shown that these
receptors are either G protein linked or ligand gated ion channels, which have wide
ranging effects on cellular function. As mentioned earlier 14 different types of 5-HT
receptors are known to date in mammalian systems. There could be new additions
to this list as 5-HT4 and 5-HT7 receptors are shown to have alternate splice variants
(see review Hoyer et al., 2002). Also recently 5-HT2 receptor is shown to have
different RNA-edited isoforms (Burns et al., 1997, also see review Niswender et al.,
1998). Now 5-HT receptors from many model organisms have been classified
based on sequence or pharmacology (Monasoratti, 1999; Tierney et al., 2001). The
Drosophila genome has been shown to have four 5-HT receptors named 5-HT1Adro
5-HT1Bdro 5-HT2dro 5-HT7dro (Saudou et al., 1992; Witz et al., 1990; Colas et al.,
1999).
One particular receptor subgroup that has interested many researchers in
vertebrate models is the 5-HT2 receptor family containing 5-HT2A, 5-HT2B and 5-
HT2C. These receptors are involved in many physiological functions like smooth
muscle contraction, feeding behavior, sleep, mood, pain, learning and memory (Roth
et al., 1998). Also 5-HT2 receptors are of interest as these receptors are targets for
many psychoactive drugs and drugs of abuse (Roth et al., 1998; Aghajanian and
Marek, 1999).
13
Depending on the levels of the agonists as well as antagonists, receptors can
undergo up- and down-regulation by alteration of their expression levels and/or by
changing their densities on the cell surface (Azaryan et al, 1998). These receptors
are regulated by altered cellular activity and developmental times. Also G protein
receptor kinases are involved in receptor desensitization, which occurs in the
presence of agonist like 5-HT and even antagonist (Hanley and Hensler, 2002).
MANIPULATIONS IN NEUROMODULATOR SYSTEMS ON FUNCTION
The role of the neuromodulators on the nervous system function,
development and whole animal development is not fully known. It is likely that an
alteration in the levels of these neuromodulators during the development are very
critical not only for development but also maintenance of neural circuits. Exact
amounts of these neuromodulators during the development are likely critical and
either higher or lower levels might result in abnormalities in the development of an
organism. Decreased levels of 5-HT in prenatal rats have shown abnormalities in
formation of different layers of neocortex in differentation and development of
neurons (Khozhai and Otellin, 2006). In Down’s syndrome (DS; trisomy 21), 5-HT
levels were shown to be lower in postmortem brains (Mann et al., 1985; Whitaker-
Azimitia; 2001). When compared to normal developing brains, DS brains have
higher levels of 5-HT1A receptor and by birth these levels drop below normal (Bar-
Peled et al., 1991). Recently Gulesserian et al. (2002) showed in adult DS patients,
serotonin transporter (SERT) levels are higher in frontal cortex. Many seretonergic
14
agents have been used in the treatment of DS, particularly to help self-injurious
behavior and aggressive behaviors (Gedye, 1990; Gedye, 1991).
Usage of certain drugs before or during pregnancy in vertebrates can result in
altering the physiological concentrations of neuromodulators, which may lead to
abnormalities in the development of a fetus or the child. For example, cocaine, a
major drug of abuse, which blocks the reuptake of dopamine and 5-HT at synapses
(Woolverton and Johnson, 1992; Filip et al., 2005), causes an increase in
cardiovascular toxicity in pregnancy. Maternal complications of cocaine ingestion are
premature labor, placental abruption, uterine rupture, cerebral ischemia and death.
Cocaine can rapidly diffuse across placenta to the fetus and cause severe
vasoconstriction. Cocaine use in pregnancy causes subtle molecular and behavioral
effects on fetal brain tissue. In postnatal life these effects are manifested in
decreased IQ scores and learning deficiencies (Krzysztof 2003). Also recently Bae
and Zang (2005) have shown that exposure of neonatal rats to cocaine causes
apoptosis and hypertrophy of myocytes in postnatal heart. Fetus exposed to cocaine
through a mother also effects the development of the brain. Due to the increased
levels of 5-HT, serotonergic terminals are not formed properly (Whitaker-Azmitia,
1998). The serotonergic system is important during the development of the
vertebrate brain.
Other prevalent drugs of abuse are amphetamines. These are a group of
non-catecholamines that produce powerful stimulation and have a prolonged activity
in the body (Krzysztof 2003). Methamphetamine is the most commonly abused type
of amphetamine. Another drug of abuse is MDMA (3,4
15
methylenedioxyamphetamine), an analog of methamphetamine. MDMA is shown to
have an effect on the developing fetus in various animal models. MDMA is known to
decrease the embryonic motility in chicken embryos. (Lyles and Cadet, 2003). In 11-
20 day old neonatal rats MDMA exposure (5-20 mg/Kg s.c, 2X/daily) causes dose-
related impairments in sequential learning and memory (Lyles and Cadet, 2003).
This time period of neonatal rats correspond to the late human trimester brain
development (Lyles and Cadet, 2003).
MDMA was used in 1970’s by psychiatrist in treating depressed patients.
Patients when given this drug would be more open in discussing their problems.
However MDMA was declared as an illegal drug by US government, so therapeutic
usage was stopped. In 1990’s MDMA became famous among teenagers as a party
drug termed “Ecstasy”. The long-term effects of MDMA assessed in rats, mice and
humans, are depletion of 5-HT and DA from neurons. MDMA can induce
neurotoxicity and cell death. In rats MDMA causes acute release of 5-HT from its
stores, which would activate the 5-HT2A and 5-HT2C receptors on the GABA
interneurons, decreasing GABAnergic transmission and increasing the DA release
and synthesis (Zhou et al, 2003). The excessively released DA can be transported
into already depleted 5-HT terminals, at the same time excessive DA is metabolized
by MAO within 5-HT terminals resulting in the excessive generation of free radicals
and reactive oxygen species (Zhou et al, 2003). There is additional evidence, which
supports that MDMA-induced neurotoxicity might occur because of the production of
superoxides rather than hydroxyl radicals (Green et al., 2003).
16
There are various effects noted in humans by exposures to MDMA such as
hallucinations, hypernatremia, hyperkalemia, psycho-stimulation, and long-term
neuropsychiatric behaviors, such as depression and psychosis (Simantov, 2004).
High doses (average of 1.04mg/L of blood) in humans results in death. In spite the
commonality of this drug and all the data that is present in the literature the specific
mechanism of action is not known. The popular model for MDMA’s mechanism of
action is through reversing the 5-HT transporter on the presynaptic nerve terminals
increasing the amount of 5-HT within the synapse until the nerve terminal is depleted
of 5-HT.
Because of all the effects of MDMA on different neurotrasmitter systems
especially on 5-HT and DA, it is possible that a fetus is developmentally affected.
Some initial studies have shown that prenatal exposure of MDMA does not effect the
development or behavior in rats (Colado et al., 1997). But a recent study in rats
showed that perinatal exposure of MDMA has led to some developmental defects in
learning and memory (Broening et al., 2001) and enhanced locomotor acitivty in later
life (Koprich et al., 2003).
Since it has proved to be difficult in the intact vertebrate brain to fully
understand the developmental consequences in neural circuits and responsiveness
of 5-HT to neurons exposed to MDMA, I chose to use a more favorable system, the
fruit fly. For several reasons the fruit fly can serve as a useful model. Drosophila, a
genetically favorable system is widely used to study the role of neuromodulators and
various studies have used flies as a model organism for the study of drugs of abuse
(Rothenfluh and Heberlein, 2002; Willard et al., 2006). Using Drosophila one can
17
relate rapidly the role of neuromodulators in the development of neural circuits and
effects on behavior. In addition the effects of MDMA on the development can be
addressed in conjugation with potential perturbations in the neuromodulators,
because it is an easy system to conduct pharmacological manipulations and
introduce mutations. Chapter 3 gives details of effects of MDMA on Drosophila larval
development, behavior and physiology. Also comparisons on effects of MDMA and
p-CPA on Drosophila larvae are reported.
5-HT Receptors and Expression in Drosophila
Another means of examining the effects of the serotonin-ergic system on
development and behaviors is not to target the biosynthesis of 5-HT but to alter the
receiving end of the 5-HT, such as the receptors. Agonists and antagonists of
various 5-HT receptors are commonly used to treat human disorders. For example,
selective serotonin reuptake inhibitors (SSRI) like fluoxetine, clomipramine are used
in the treatment of autism (Hollander et al., 2003; Namerow et al., 2003), 5-HT3
antagonist alosetron is used in the treatment of irritable bowel syndrome (IBS) in
females (Andresen and Camilleri, 2006), atypical anti-psychotic drugs in the
treatment of schizophrenia (Meltzer at al., 2003; Stimmel et al., 2002). It is known
that people with altered levels in expression of particular 5-HT receptors can show
social and mental deficits (Whitaker-Azmitia, 2001; Sodhi and Sanders-Bush; 2004).
Perhaps the lack of the appropriate 5-HT receptor expression throughout neural
development is the cause for a number of aliments in humans that have yet to be
correlated to molecular mechanisms. There are various polymorphisms in races of
people for 5-HT receptors which are noted to be responsible for differential effects to
18
drug therapies (Bolonna et al., 2004; Reynolds et al, 2005). This is a growing
interest of pharmaceutical companies as well as medicine in general in order to
provide therapy based on one’s genomic identity (Bolonna et al., 2004; Reynolds et
al, 2005).
This emerging field of study in mammals is exciting for many reasons. One
being that it will help to understand the interaction of receptor expression and more
specific drug therapies to reduce side effects from broad spectrum agonists and
antagonists but in time there will be more interest in the developmental
consequences in slight to extreme modifications to particular neural systems, like the
neural circuitry that is impacted by 5-HT modulation. This is one reason why I
pursued the potential effects on development in Drosophila related to the alteration
in the appropriate expression of 5-HT receptor subtypes.
Genomic analysis has shown that there are four receptor types for 5-HT in
Drosophila (Witz et al., 1990; Saudou et al, 1992; Colas et al, 1994; Tierney, 2001;
Peroukta; 1994). As mentioned earlier they are named as 5-HT7dro, 5-HT1Adro, 5-
HT1Bdro and 5-HT2dro based on sequence and functional similarities with the
mammalian 5-HT receptors 5-HT1A, 5-HT2 and 5-HT7. Nichols et al (2002) showed
that LSD in flies may be mediating its affects through 5-HT1Adro and 5-HT2dro. In
general very little work has been conducted on Drosophila 5-HT receptors. The 5-
HT2dro is 40% homologous over the transmembrane domain of 5-HT2 receptor of
mammals. 5-HT2dro is present on 3rd chromosome and right arm. Two transgeneic
lines have been made concerning this receptors that I have taken advantage of in
my studies. One with an anti-sense strand of the gene under heat-shock promoter,
19
called Y32 and another with an anti-sense strand under heat shock Gal4-UAS
system. In chapter 4, I report on 5-HT2dro role in Drosophila development, behavior
and physiology.
Using the Drosophila heart as a bioassay for 5-HT effects
Since I have focused on the role of 5-HT in CNS function and behavioral
studies I wanted to use an additional physiological assays for examining the holistic
effects of the serotonin-ergic system. During the progression of the dissertation
studies I had become aware that the heart of insects and crustaceans is very
susceptible to exogenous application of 5-HT. In fact, students in the laboratory were
using the heart rate as a bioassay for social interactions in crayfish with the notion of
testing if there was a correlation to aggressive and submissive roles (Listerman et
al., 2000). The underlying assumption was that if aggressive individuals have a
higher level of circulating 5-HT, as proposed in earlier studies (Livingston et al.,
1980), then the aggressive animals should have a higher heart rate as compared to
submissive ones. Investigating actions of 5-HT on Drosophila hearts I discovered
that studies had been conducted in Drosophila. However, I also discovered some
shortcoming in the past procedures used to examine the actions of 5-HT on the
heart of larval Drosophila.
Thus in Chapter 5, I present a full study that has already been published on
the effects of 5-HT to the exposed larval heart with and without an intact CNS. The
regulation of the heart via hormonal and direct neural innervation had been
conducted primarily in adult hearts (Dulcis and Levine 2003, 2005; Dulcis et al.
2005; Johnson et al. 2002; Miller, 1997; Papaefthimiou and Theophilidis, 2001).
20
However only recently using GFP expressing lines of flies was this investigated in
larvae by a fellow student in the laboratory, Dr. Andrew Johnstone. Since my
findings presented in chapter 5 indicated that there are differences in heart rate
depending if the CNS is intact or not, a study was conducted to examine possible
connections from the CNS to the heart. They found nerves from the CNS leading to
the dorsal aorta and in electron micrographs nerve terminals containing synaptic
vesicles, thus suggesting direct motor nerve regulation of the heart (Johnstone and
Cooper, 2006).
It has been known for some time that the heart rate in larvae can be altered
by neurotransmitters and neuromodulators, which are known to be present in the
hemolymph (Johnson et al. 1997, 2000; Nichols et al. 1999; Zornik et al. 1999). This
was primarily examined up by injections in 3rd instars and early pupa (P1 stage,
transition between larva and pupa) of 5-HT, DA, Ach, octopamine (OA), and
norepinephrine (NE) which all increase HR (Johnson et al. 1997). Injection of 5-HT
(1�M/l) caused the HR to increase by 46% from base line (Johnson et al. 1997) and
Zornik et al., (1999) showed, in the wandering 3rd instar larva, that 5-HT increases
HR by 111% with a concentration of 10-5 M (10�M/l).
The thought that injection through the larval body wall or into a pupal case of
biogenic amines can cause the activation or release of many other compounds
struck my interest. This did not seem to draw attention by past investigators to
control during the experiments. My thought was that even saline injection could
induce stress and potential release of 5-HT. Thus, I wanted to try direct application
on exposed hearts in a defined saline. The ability to investigate the sensitivity of the
21
heart to 5-HT also was of interest since I could use the preparation as a bioassay to
the sensitivity to 5-HT in the studies in which the levels of 5-HT had been reduced by
feeding larval p-CPA and MDMA. Additionally, this heart bioassay would serve of
interest to the studies in which I was using the fly strains that had a suppressed
expression of the 5-HT2 receptors (Chapter 4). Considering there were many
avenues in which the larval heart bioassay to 5-HT was going to be of use to my
other studies I decided to do a complete investigation on the subject. The findings
presented in Chapter 5 served as a baseline to compare results in the other studies
related to 5-HT production (Chapter 3) and altering 5-HT receptor expression
(Chapter 4). Also since MDMA has direct action on neuronal 5-HT receptors I
continued studies with MDMA to examine potential direct action on the larval heart in
order to parallel the 5-HT study on the CNS.
The specific aims of this dissertation research are:
1) Address the role 5-HT in the development of Drosophila and changes in
central nervous system physiological response due to pharmacological
manipulations (by p-CPA or MDMA) during the development.
2) Determine effects of MDMA on development and physiological response of
central nervous system.
3) Address effects caused by the lack of the major receptor (5-HT2dro) on
development and responsiveness of the larval CNS to exogenous 5-HT,
MDMA application.
Addressing these aims are very important in understanding processes in
neuronal develop in relation to whole animal behavior and the impact of
22
neuromodulators. 5-HT as described earlier is an important molecule in the
development of brain and whole animal. To dissect out the role 5-HT plays in
mammals is difficult due to the complexity. Hence using a simpler organism,
Drosophila melanogaster, is advantageous and the results obtained here can be
extrapolated to higher organisms. I have shown that 5-HT and its receptor plays a
vital role in the development of Drosophila and its physiology.
Copyright © Sameera Dasari 2007
23
CHAPTER 2
MODULATION OF SENSORY-CNS-MOTOR CIRCUITS BY SEROTONIN, OCTOPAMINE, AND DOPAMINE IN SEMI-INTACT DROSOPHILA LARVA
ABSTRACT
I have introduced an in-situ preparation to induce motor unit activity by
stimulating a sensory-CNS circuit, using the 3rd instar larvae of Drosophila
melanogaster. Discrete identifiable motor units that are well defined in anatomic and
physiologic function can be recruited selectively and driven depending on the
sensory stimulus intensity, duration, and frequency. Since the peripheral nervous
system is bilaterally symmetric to coordinate bilateral symmetric segmental
musculature patterns, fictive forms of locomotion is able to be induced. Monitoring
the excitatory postsynaptic potentials on the prominent ventral longitudinal body wall
muscles, such as m6 and m12, provides additional insight into how the selective
motor units might be recruited within intact animals. We also introduce the actions
of the neuromodulators (serotonin, octopamine and dopamine) on the inducible
patterns of activity within the sensory-motor circuit. The powerful genetic
manipulation in Drosophila opens many avenues for further investigations into the
circuitry and cellular aspects of pattern generation and developmental issues of
circuitry formation and maintenance in the model organism.
24
INTRODUCTION
Sensory input early in life sculpts central circuits, which can become relatively
hard wired after defined critical periods. This was most elegantly shown in the 1960's
experimentally for the visual system in cats and monkeys (Hubel and Wiesel, 1963a,
b, 1968, 1970) and is clinically relevant to humans. Other parts of the brain also
show similar dependences on sensory activity in development. The formation of
cortical circuits is of interest since this controls thought processes and forms of
learning (Pallas, 2001). Refined experimentation of sensory attributes defining CNS
and motor units have been possible in relatively less complex organisms. A striking
example is in the development of the asymmetric claws of lobsters (Lang et al.,
1978) where Govind and colleagues demonstrated that juvenile lobsters depend on
sensory stimulation for the asymmetry to occur (Govind and Pearce, 1986). When
lobsters (Homarus americanus) are not allowed to manipulate objects in their claws
they will develop two cutter claws, where as if one claw is exercised a crusher claw
will develop over subsequent molts for the side that had prior enhanced sensory
stimulation. Not only is the muscle phenotype, biochemistry, and cuticle
differentiated but the number of sensory neurons and the central neuropile in the
thoracic ganglion are modified during development of the asymmetry (Cooper and
Govind, 1991; Govind and Pearce, 1985; Govind et al., 1988).
In the genetically favorable invertebrate Drosophila, Suster and Bate (2002)
produced embryos with reduced sensory function, which results in abnormal
peristalsis of embryonic movements, which suggests sensory activity is
developmentally important in shaping central control of motor output within
25
invertebrates. However, the problem still challenging the field is in understanding the
integration of sensory input that controls muscular movements in a coordinated
fashion. Recent studies in pharmacological treatments of spinal cord injuries in cats
and in humans have revealed that recovery of locomotion is enhanced by using
selective agonists and antagonist of neurotransmitters involved in sensory-CNS-
motor circuits (Chau et al., 2002; Rossignol, 2000; Rossignol et al., 2001, 2002).
These recent studies are a breakthrough in manipulating selective sensory systems
and higher order function in controlling motor output.
The ability to combine a genetically favorable system and pharmacological
studies is opening new horizons in regulation of development in neural circuits. In
addition, neuromodulators provide a rapid way in which animals can tune up or down
activity within a neural circuit and may be responsible for rapid changes in behavior,
as recently examined for aggressive behavior in Drosophila (Baier et al., 2002). We
assessed three common neuromodulators of interest in arthropod neurobiology:
serotonin (5-HT), octopamine (OA), and dopamine (DA). Voltage dependent
potassium channels and heart rate are modulated by 5-HT in Drosophila (Johnson et
al., 1997; Zornik, 1999). DA is known to alter sexual behavior, habituation
(Neckameyer, 1998a, b) and increase activity in adult flies (Friggi-Grelin et al., 2003)
but depress synaptic transmission at the NMJ in larval Drosophila (Cooper and
Neckameyer, 1999). Behaviors in bees are also affected by DA (Taylor et al., 1992).
OA expression is related to stress responses in Drosophila (Hirashima et al., 2000)
and OA receptors are present in mushroom bodies in Drosophila CNS (Han et al.,
1998). These past studies indicate that there is a precedence of 5-HT, DA, and OA
26
to have central effects in the Drosophila brain (Baier et al., 2002; Blenau and
Baumann, 2001; Monastirioti, 1999). The purpose of these studies is present an in
situ preparation of larval Drosophila, with intact sensory-CNS-motor circuits, to serve
as a model system for investigating actions of neuromodualtors on developing
central circuits.
METHODS
Many of the procedures used here have been previously described in detail
(Ball et al., 2003; Cooper and Neckameyer, 1999; Li and Cooper, 2001; Li et al.,
2001, 2002). The staining of the nerve terminals with an antibody to HRP was
described previously (Li et al., 2002). In brief, the following procedures and condition
were used with the modifications emphasized.
Stock and Staging of Larvae
The common ‘wild-type’ laboratory strain of Drosophila melanogaster, Canton
S, was used in these studies. The methods used to stage fly larvae have been
described previously (Campos-Ortega and Hartenstein, 1985; Li et al., 2002). Larvae
at the beginning of the “wandering” phase of the third instar were used in these
experiments.
Dissection and physiological conditions
Dissections included removal of the heart and viscera which left a filleted
larvae containing only a body wall, body wall muscles and the neural circuitry for the
sensory, CNS and body wall (i.e., skeletal) motor units as described earlier (Cooper
27
et al., 1995). The HL3 saline was prepared in the lab from component reagents
(Sigma) and contained: 1.0 mM CaCl2.2H2O, 70mM NaCl, 5mM KCl, 10mM
NaHCO3, 5mM trehalose, 115mM sucrose, and 5mM BES (N,N-bis[2- Hydoxyethyl]
-2-aminoethanesulfonic acid) (Stewart et al., 1994).
Electrophysiology
The recording arrangement was essentially the same as previously described
(Neckameyer and Cooper, 1998; Stewart et al., 1994). Intracellular recordings in
muscles were made with 30-60M� resistance, 3M KCl-filled microelectrodes. The
amplitudes of the excitatory postsynaptic potentials (EPSP) elicited by Is and Ib
motor nerve terminals in the various segments of muscles m6 and m12 were
monitored. Intracellular responses were recorded with a 1 X LU head stage and an
Axoclamp 2A amplifier. Stimulation of segmental nerve roots was provided by
suction electrodes (Cooper and Neckameyer, 1999). The stimulator (S-88, Grass)
output was passed through a stimulus isolation unit in order to alter polarity and gain
(SIU5, Grass). Electrical signals were recorded on-line to a PowerMac 9500 and
G4 Mac via a MacLab/4s interface. All events were measured and calibrated with
the MacLab Scope software 3.5.4 version. All experiments were performed at room
temperature (19-22oC).
RESULTS
In filleted 3rd instar larvae, each segmental nerve root and ventral body wall
musculature is readily observed (Fig. 2.1A). Various identified muscles with a rather
simplistic innervation profiles can be used to monitor motor neuron activity (Fig.
28
2.1B). In these studies, we utilized muscle 6 (m6) and muscle 12 (m12) because of
the well characterized innervation and synaptic properties of the Is and Ib motor
nerve terminals (Fig. 2.1C) (Atwood et al., 1993; Kurdyak et al., 1994; Li et al.,
2002). Each segmental nerve root can be stimulated to drive sensory input into the
larval brain as well as stimulating motor neurons to the segmental muscles that
particular root is associated. By transecting the root and only stimulating the distal
aspect of the root, the motor neurons are devoid of CNS activity and defined
patterns of stimulation can be given. Likewise, the proximal root can either be left
intact or transected to drive sensory patterns to the CNS for a particular segment or
segments when multiple roots are utilized. Here we used single intact segmental
roots to drive central circuits and record motor unit activity in contra-lateral and ipsi-
lateral segments to the segment being stimulated (Fig. 2.1B).
Since the innervation to m6 and m12 is well defined, one can assess which
specific motor neurons are being recruited as a result of sensory stimulation by
monitoring the EPSPs induced in these particular muscles. The responses that can
be evoked in m6 in the various segments when stimulating the 3rd segmental nerve
on the right side is shown in Figure 2.2A. When monitoring two muscles
simultaneously, selective motor neurons that are recruited which innervate both m6
and m12 or motor units which exclude m12 are able to be observed (Fig. 2.2B). In
addition, since the Ib and Is motor nerve terminals that innervate m6 show different
morphology and physiological responses they can be discerned individually or when
they are recruited in unison. The terminals of the Is axon contain small varicosities
along its length and give rise to large EPSPs in the muscle, where as the Ib axon
29
has big varicosities on its terminals (Fig. 2.1C and 2.2C), but produces smaller
EPSPs (Atwood et al., 1993; Kurdyak et al., 1994; Stewart et al., 1994). The induced
depolarizations on these muscles are graded and are non-spiking.
To examine if recruitment of sensory axons, interneurons and motor neurons
is dependent on stimulation, three stimulation conditions were used. First, we
examined the response of the motor units to stimulus duration. An increase in the
duration of a train of stimuli enhanced activity of motor units (Fig. 2.3A, 40Hz with 10
stimuli; B, 40Hz with 15 stimuli). In addition, increasing the frequency of stimulation
recruited motor units rapidly as compared to lower stimulus frequencies (Fig. 2.3A,
40Hz with 10 stimuli; C, 60 Hz with 10 stimuli). The amount of motor activity is also
dependent on the intensity of stimulation (Fig. 2.3D, 40Hz with 10 stimuli low
stimulus voltage). In 5 out of 5 preparations, the higher the stimulation frequency
(40 Hz to 60 Hz), the longer duration of the stimulation (10 pulses to 20 pulses at 40
Hz), and the higher the stimulation intensity (increased by 1 V to the stimulating
electrode) all resulted in an increase in the average activity of the motor neuron. The
percent change from 40 Hz with a 10 pulse train is used for comparison (Fig. 2.3E).
For this analysis, five periods of 500 msec duration, every ten seconds, were
obtained and an average number of EPSPs was determined. Increasing the
stimulation duration had the greatest effect in enhancing motor unit activity. It should
be noted if the stimulation intensity is too large a failure to evoke action potentials
could occur. Thus, some sensory neurons may drop out of as stimulation increases
to very large voltages.
30
To determine the effects of 5-HT, OA, and DA in altering the sensory to motor
neuron central circuit a segmental root was stimulated while the evoked responses
in the contra-lateral m6 were monitored prior and during exposure to
neuromodulators. The neuromodulators were applied by rapidly exchanging the
entire bathing media with a saline containing the desired concentration. A single
preparation was used for a given manipulation. Since the degree of recruiting motor
neurons varied in each preparation a percent difference in the firing frequency of the
motor units was quantified (Fig. 2.4A). OA at 10�M resulted in massive waves of
muscle contraction making it difficult to maintain an intracellular recording (n=6).
Thus, a lower concentration of 1�M was used for OA as compared to 5-HT and DA.
In all cases, OA enhanced the firing frequency of the motor units. 5-HT (10�M)
showed biphasic effects in altering the frequency of evoked motor unit response.
Initially an enhancement in the frequency was observed but within 1 to 2 minutes a
decrease in the frequency of the evoked responses occurred. The frequency in the
evoked responses was measured for the peak excitatory effect within the first minute
and the frequency after 2 minutes. The results are shown for 5 preparations (Fig.
2.4A). Only a small excitatory effect was observed for DA (10�M), however like for
5-HT, a transitory effect was observed (See enlarged inset). Five preparations were
used for each compound and in each case the direction of change was the same
(p<0.05, n=5, non-parametric rank sum Wilcoxon test). To illustrate the biphasic
response induced by 5-HT the first stimulus train and resulting EPSPs after
exposure is shown (Fig. 2.4B1) along with a stimulus train 1 minute and 26 seconds
later (Fig. 2.4B2).
31
In examining the direct effects of the neuromodulators at the NMJ, a
transected segmental nerve was stimulated distally to evoke a combined response
from the Ib and Is terminals on m6. A percent change in the amplitude of the
composite EPSPs revealed that both OA and DA reduced the amplitude (p<0.05,
non-parametric rank sum Wilcoxon test) where as 5-HT had no significant effect on
the response (Fig. 2.4C).
DISCUSSION
The topic of rhythmic control of locomotion is an age old question since
Sherrington's time (Sherrington, 1898). Significant breakthroughs have occurred
over the years, however the regulation and neural integration of locomotion remains
a significant hurdle for the field. In this report, I demonstrate that the model
organism, Drosophila melanogaster offers a unique advantage to begin to address
pattern generation involved in locomotion as well as the role various sensory inputs
have that drive circuits. In addition, the role of neuromodulators which is now proving
to be advantageous to alter locomotive patterns in spinal injury models in mammals
can also be assessed in larval Drosophila. The powerful genetic manipulation of the
organism opens many avenues for further investigations into the circuitry and
cellular aspects of sensory integration. The goal of this technical report is to present
one with a preparation to address physiological effects in development and
maintenance of central circuitry that could possibly be correlated with behavior.
The projections of sensory neurons in the larva can influence later
development of novel sensory neurons in the adult, thus pharmacological
32
manipulation or altered activity profiles in the larva can be examined in shaping the
adult CNS of holomotabolus insects. Targeting particularly gene mutations in
Drosophila towards specific sensory neurons or even all sensory neuronal function
by inducible tetanus toxin light chain expression (Suster and Bate, 2002) within
neurons will allow refined and gross manipulations of the circuitry for assessment of
function and adaptation. As with C. elegans (Francis et al., 2003), genetic alterations
in the expression of proteins involved in synaptic transmission result in behavioral
patterns that can be quantified in larval and adult Drosophila (Neckameyer and
Cooper, 1998; Li et al., 2001).
In our initial investigations, we were interested in monitoring fictive locomotion
from recordings of the segmental nerves in filleted and pinned larvae (Fig. 2.1).
Rhythmic patterns do appear, but the patterns are not reliable between preparations.
In addition, when bursts of activity are recorded, the frequency profiles run down
rather quickly making it difficult for long term assessment of fictive locomotion
patterns. Hence, we turned to an alternative approach of driving the motor units by
sensory nerve stimulation and then assessing the role of neuromodulators on the
circuit. A similar approach has been used in the semi-intact leech preparation where
electrical stimulation of sensory roots produces a escape swim circuit (Weeks,
1981). The fictive swimming can also be induced by exposure of the ventral nerve
cord to 5-HT (Willard, 1981). Like wise, locomotor activity in the isolated spinal cord
of the lamprey can be induced by bath application of NMDA (Svensson et al., 2003).
The stomatogastric ganglion (STG) of crustaceans also serves as a nice
invertebrate model for investigating actions of neuromodulators on motor patterns. It
33
has been shown in the STG that neural circuits and the networks are modulated by
biogenic amines and there is both convergence and divergence in their action
(Marder and Thirumalai, 2002).
Our particular interests focus on the influences of hormones and
neuromodulators in altering central circuitry, particularly the ones already known to
have a role in altering synaptic growth and plasticity at the neuromuscular junction
(Cooper and Neckameyer, 1999; Li and Cooper, 2001; Li et al., 2001; Neckameyer
and Cooper, 1998; Ruffner et al., 1999). It is well established that hormones such
as ecdysone and juvenile hormone alter neural development and differentiation in
insects (Garen et al., 1977;Pak and Gilbert, 1987; Truman, 1996). The surge of
ecdysone in the pupal stage of Drosophila likely plays a key role in inducing gross
alterations in neural circuitry (Kraft et al., 1998; Thummel, 1996; Truman and Reiss,
1988) and motor unit function (Li and Cooper 2001; Li et al., 2001). Likewise, other
hormones or cocktails of other hormones need to be investigated for their
developmental roles, since it has been demonstrated that the sequence of
neuromodulator exposure and cocktails produce differential effects on synaptic
modulation in other arthropods (i.e., the crustaceans) (Djokaj et al., 2001).
Since in the intact organism, compensatory mechanisms may override
experimentally induced genetic, hormonal or environmental alterations, one can now
turn to whole CNS and body musculature culture of larval Drosophila to address
specific questions (Ball et al., 2003). However many compounding variables need to
be considered, such as the loss of normal movements and appropriate feedback
responses in culture conditions. The physiological saline based on the composition
34
of larval hemolymph, HL3, preserves synaptic transmission as well as muscular
function and integrity (Stewart et al., 1994). Slight modifications of the HL3 saline are
used for culturing the preparation (Ball et al., 2003), but perhaps the recently
developed HL6 saline (Macleod et al., 2002) should be examined. With the
physiological method presented, genetic or pharmacological manipulation of
neuromodulators over a long-term, in the whole animal or in culture, can be readily
assessed. However, the challenge is now to determine where the neuromodulators
are acting (i.e., sensory, interneurons, and/or motor neurons) and what receptor
subtypes exists.
35
FIGURES
Figure 2.1: Schematic diagram of the Drosophila larva preparation:
(A) The preparation is pinned at the four corners to keep the preparation taut. The
ventral abdominal muscles, m6 and m12, were used in this study. (B) The
segmental nerves can be stimulated by placing the nerve into the lumen of a suction
electrode and recruiting various subsets of sensory neurons. The segmental roots
can be severed from the body wall to selectively stimulate sensory nerves
orthodromicaly. (C) The terminals of Ib and Is on m6 and m7 are readily observed
after treatment with fluorescently tagged anti-HRP antibody. Scale: 750 �m A & B,
90�m C.
36
Figure 2.2: Representative traces of induced responses recorded in muscle 6 in
various segments.
The 3rd segmental nerve on the right side of the larva was stimulated at a given
voltage and frequency while responses were monitored in the m6 (A) and m12 (B)
muscles on the contra-lateral side to the stimulated nerve root. In segment 3,
contra-lateral to the segmental root being stimulated, EPSP responses in two
different muscles m6 and m12 reveal that selective motor neurons can be recruited.
The motor neuron RP3 innervates both m6 and m12 while the motor neuron 6/7b
innervates m6 but not m12. Sometimes the Ib is selectively recruited since only a
response in m6 is observed. (C) Elicited responses in m6 is readily possible with a
37
intracellular recording as a consequence of stimulating the transected segmental
root. Representative individual responses from the Ib and Is motor axons as well as
the composite Ib and Is response are shown from late 3rd instars.
38
Figure 2.3: Recuritment of motor units. Recruitment of motor units is dependent on
the duration of the stimulation.
(A, 40Hz, 10 pulses; B, 40Hz, 15 pulses), frequency of stimulation (A, 40Hz, 10
pulses; C, 60Hz, 10 pulses), and intensity (C, 60Hz, 10 pulses high stimulus voltage;
D, 60Hz, 10 pulses low stimulus voltage). Represented are EPSPs recorded in m6
induced by stimulating the contra-lateral segmental root. At subthreshold (D)
stimulation of sensory afferents no inducible responses are observed. However,
recruitment occurs with an increased stimulation intensity (C). Stars in top trace
indicate stimulus artifacts for the first three within the stimulus train. (E) An average
percent change from 40 Hz with a 10 pulse train is used for comparison to a higher
the stimulation frequency (60Hz), a higher the stimulation intensity (increased by 1 V
39
to the stimulating electrode), and a longer duration of the stimulation (20 pulses at
40 Hz) (at least n=5 for each condition).
40
Figure 2.4: The influence of neuromodulators in altering the sensory to motor neuron
central circuit was examined.
(A) A percent difference in the firing frequency of the motor units to m6 was
determined before and during exposure to a either serotonin (5-HT,10�M),
octopamine (OA, 1�M), or dopamine (DA, 10�M). Five independent preparations
were examined for each neuromodulator. Since biphasic responses were observed
for 5-HT, a peak enhancement in the firing frequency was measured within 1 minute.
41
The peak response and an average response for 2 minutes were used for analysis.
The inset shows an enlarged view of the bar chart for the DA responses. A typical
biphasic response induced by 5-HT is depicted by comparing B1 (upon initial
exposure) to B2 (1 minute and 26 seconds later). (C) Direct assessment of OA
(1�M), DA (10�M), and 5-HT (10�M) on the amplitude of evoked combined Is and Ib
EPSPs at the neuromuscular junction on m6 revealed that both OA and DA
depressed synaptic transmission.
42
CHAPTER 3
INFLUENCE OF P-CPA AND MDMA ON THE SEROTONERGIC SYSTEM IN RELATION TO PHYSIOLOGY, DEVELOPMENT AND BEHAVIOR OF
DROSOPHILA MELANOGASTER
ABSTRACT
Biogenic amines like serotonin (5-HT) are known to have a role in
development and behavior. In this study the serotonergic system was altered using
para-chlorophenylalanine (p-CPA) in order to study its role on development,
behavior and physiology in larval Drosophila. Since MDMA is known to deplete 5-HT
in neurons in mammals parallel studies to p-CPA were conducted. p-CPA and
MDMA delayed time to pupation and eclosion. Locomotion and eating were reduced
in animals exposed to these compounds. Sensitivity to exogenously applied 5-HT on
a evoked sensory-CNS-motor circuit showed that the CNS is sensitive to 5-HT but
that when depleted of 5-HT by p-CPA no enhanced sensitivity was observed. Larvae
eating MDMA from 1st to 3rd instar did not show a reduction in 5-HT within the CNS;
however, eating p-CPA reduced not only 5-HT but also dopamine content. Since the
heart serves as a good bioindex to 5-HT exposure, it was used in larva fed p-CPA
and MDMA, but no significant effects were noted to exogenously applied 5-HT in
these pharmacologically treated larvae.
INTRODUCTION
Serotonin (5-HT), dopamine (DA) and octopamine (OA) are well known to act
as neuromodulators in insects, particularly in Drosophila melanogaster, which when
altered can produce behavioral and developmental defects as well as organizational
43
problems in the CNS circuits (Monastirioti, 1999; Osborne, 1996). 5-HT modulates
voltage dependent potassium channels and heart rate in Drosophila (Johnson et al.,
1997; Zornik et al., 1999). DA is known to alter sexual behavior, sensory habituation
(Neckameyer, 1998a,b) and increase activity in adult flies (Friggi-Grelin et al., 2003)
but depress synaptic transmission at the NMJ in larval Drosophila (Cooper and
Neckameyer, 1999). OA expression is stress related in Drosophila (Hirashima et al.,
2000) and OA receptors are present in mushroom bodies in Drosophila CNS which
is a region important for learning in adults (Han et al., 1998). In fact, 5-HT, DA, and
OA all have some central effects in the adult Drosophila brain related with learning
or behavior (Blenau and Baumann, 2001; Monastirioti 1999). Recently, direct
actions of these neuromodulators were shown to alter central neural activity (Dasari
and Cooper, 2004).
These biogenic amines have broad differential effects on development and
physiology in larvae as well as in adults. Here I focus on the serotonergic system
and tissue sensitive to alterations in endogenous levels of 5-HT within larvae and
pupa. The development and the distribution of 5-HT immunoreactivity neurons in the
CNS are established (Valles and White, 1988). 5-HT has a role in many
physiological process such as regulating locomotion and cardiac output (Dasari and
Cooper, 2006; Kamyshev et al., 1983; Johnson et al., 1997; Nichols et al., 1999;
Zornik et al., 1999; Johnson et al., 2000). Since 5-HT alters the activity of sensory-
to-motor central circuits in larval Drosophila (Dasari and Cooper 2004) this opens
the possibility that the serotonergic system could sculpt the formation of neural
circuits by altering the neural activity in the developing CNS of Drosophila. Activity of
44
developing neural circuits is well established to play a major role in the patterning of
the adult CNS in mammals prior to critical periods (Hubel and Wiesel, 1963a,b,
1968, 1970).
MDMA (ecstasy), a drug of abuse, modulates the homeostasis of the
serotonergic system in humans and animal models (Green et al., 2003). Research is
scant on the effects of MDMA in the developing CNS of mammals as well as in
insects. Thus, I used the rapidly developing nervous systems of Drosophila larvae to
provide an avenue to quickly screen the effects of MDMA on the larval CNS. The
proposed mechanism of MDMA's action in mammals is an eventual depletion of 5-
HT within neurons. I tested other means, through pharmacological manipulation,
throughout early stages of larval development to deplete 5-HT for comparison with
the effects induced by MDMA.
METHODS
Stock and Staging of Larvae
The common ‘wild-type’ laboratory strain of Drosophila melanogaster, Canton
S, was used in these studies. The methods used to stage fly larvae have been
described previously (Campos-Ortega and Hartenstein, 1985; Li and Cooper, 2002).
All animals were maintained in vials partially filled with a cornmeal-agar-dextrose-
yeast medium. All animals were kept on a 12:12 light-dark cycle. Introduction of
pharmacological agents started with the 1st instar.
Behavioral assays
45
Early 3rd instar larvae were used for behavioral assays. Feeding and
locomotory behavior was assessed as described in Neckameyer (1996) and Li et al.
(2001). In brief, single animals were placed on a 2% agar surface and the number of
body wall contractions was counted for 1 minute, after which an animal was placed
in a 2% yeast solution overlaid on an agar plate (just covering the larvae with
allowing the spiracles to reach out of the solution). In this condition, Drosophila
larvae immediately feed, initiating a pattern of repetitive mouth hook movements.
The number of full mouth hook contractions in 1 minute was counted (Sewell et al.,
1975). The results of these behaviors are plotted as body wall contractions or mouth
hook movements per minute.
Dissection and electrophysiological recordings
Wandering 3rd instar larvae were dissected as described earlier (Cooper et
al., 1995). In brief larvae were dissected dorsally removing heart and viscera which
left a filleted larvae containing only the body wall, body wall muscles and the neural
circuitry for the sensory, CNS and body wall (i.e., skeletal) motor units. HL3 saline
was prepared in the lab from component reagents (Sigma) and contained: 1.0 mM
CaCl2·2H2O, 70 mM NaCl, 20 mM MgCl·6H2O, 5 mM KCl, 10 mM NaHCO3, 5 mM
trehalose, 115 mM sucrose, and 5 mM BES (N,N-bis[2-Hydoxyethyl]-2-
aminoethanesulfonic acid) (Stewart et al., 1994).
The recording arrangement was essentially the same as previously described
(Neckameyer and Cooper, 1998 and Stewart et al., 1994). Intracellular recordings in
muscles were made with (30–60 M resistance), 3 M KCl-filled microelectrodes. The
amplitudes of the excitatory postsynaptic potentials (EPSP) elicited by Is and Ib
46
motor nerve terminals of muscle m6 were monitored. Primarily body segments 3 and
4 were used throughout these studies. Intracellular responses were recorded with a
1×LU head stage and an Axoclamp 2A amplifier. To evoke a sensory-CNS-motor
circuit the tail segmental nerves were cut and stimulated using the suction electrode
while using an intracellular electrode in a m6 muscle fiber (Dasari and Cooper 2004).
The stimulator (S-88, Grass) output was passed through a stimulus isolation unit in
order to alter polarity and gain (SIU5, Grass). Electrical signals were recorded on-
line via an A/D converter (powerlab 4s interface; ADInstruments). All events were
measured and calibrated with Scope software 3.5.4 version (ADInstruments). All
experiments were performed at room temperature (19–21 °C).
Developmental Assays Eggs were collected after a 15-minute prepulse for 2 hrs and allowed to
develop at 21oC. Eggs were transferred to vials (15/vial) containing food with p-CPA
or MDMA so that 1st instars are in the food right after hatching. The food was made
with 0.5 g of corn meal with 0.5 ml of water mixed with the appropriate concentration
of drug. I used corn meal instead of yeast-water for the food since we found a high
rate of death with the yeast paste. The deaths were probably due to CO2 build up
with the growth of the yeast. When wandering 3rds were seen, the vials were
checked every 4 hrs and each individual pupa was marked on the side of the vials.
The time to pupation and the time spent as a pupa were indexed for development.
47
Heart Rate
Heart rate in 3rd instar was examined and monitored in the same manner as
detailed in Dasari and Cooper (2006).
Levels of Serotonin and Dopamine The 1st instars were fed with pharmacological agents as described above.
The wandering 3rd instars were used for selective measures on the CNS. Larvae
were dissected in HL3 as described above. Brains were placed in dry ice
immediately after dissecting. 25 brains were pooled for each sample set and stored
in –80oC prior to HPLC analysis. HPLC analysis was performed at The Center for
Sensor Technology (University of KY, USA).
The low level detections of 3-Hydroxytyramine (Dopamine; DA) and 5-
Hydroxytryptamine (Serotonin; 5-HT) are performed as described earlier by Hall et al
(1989). In brief an isocratic HPLC system (Beckman, Inc., Fullerton, CA), at a flow
rate of 2 ml/min., which is coupled to a dual-channel electrochemical array detector
(model 5100A, ESA, Inc., Chelmsford, MA), E1 = +0.35 mV and E2 = -0.25 mV, using
an ESA model 5011 dual analytical cell. The compounds of interest are separated
with reverse-phase chromatography, using a C18 column (4.6 mm x 75 mm, 3 µm
particle size, Shiseido CapCell Pak UG120, Shiseido Co., LTD., Tokyo, Japan) with
a pH 4.1 citrate-acetate mobile phase, containing 4.0% methanol and 0.34 mM 1-
octane-sulfonic acid.
48
Statistical Analysis When the basic assumption of parametric Student's-t test was valid it was
used; otherwise, the non-parametric Wilcoxon rank sum test was used.
RESULTS
Developmental curves
The first set of general analysis was to examine the time it took 50% of the
population to pupate. The time for 50% of the population to reach eclosion from
pupation was also assessed. p-CPA or MDMA at various concentration was fed to
larvae throughout 1st to 3rd instar stages. The time taken by each larva from egg
(Time=0) to pupation was recorded. The highest concentration of p-CPA (50mM;
10mg/ml) showed a drastic delay in the development such that 50% of larvae took a
mean time of ~225 hrs for pupation, whereas 50% of controls pupated at ~125 hrs
(Fig. 3.1A, B). There was a high death rate in this group (64% + 5.8). There were a
small number of deaths in controls (2% + 2) and at lower concentrations of p-CPA
treated larvae. Larvae fed p-CPA at 50mM were generally smaller in size on average
as compared to controls (this was based on casually observation and not statistically
analyzed). p-CPA at 0.5 mM showed no delay from controls in the time for 50% of
animals to pupate and 5 mM showed only a slight delay of 35 hrs from controls for
pupation time (Fig. 3.1A, B). In calculating the amount of time taken for each pupa to
eclose the time point at which pupa a formed was readjusted to a zero time point
(Time=0). The time taken for p-CPA (50mM) fed larvae to eclose is longer than for
controls (>35 hrs, Fig. 3.1C, D). They seem to catch up in eclosion time even though
49
there is delay in pupation time. A similar result was also seen with the other
concentrations of p-CPA (Fig. 3.1 C, D).
Similar results to p-CPA were seen with MDMA fed larvae. MDMA at highest
concentration (1mM) showed a delay in time to pupation by approximately 36 hrs for
50% of larvae to pupate (Fig. 3.2A, B). A 40% (+ 7) death rate was seen in this
group. The lower concentration 10 �M MDMA fed larvae took less time (10 hrs) for
50% animals to pupate compared to controls and 100 �M MDMA took ~10 hrs more
than controls for 50% of the animals to pupate (Fig. 3.2A, B). Eclosion time for 1mM
fed larvae is about 20 hrs more than the controls, whereas the other 2
concentrations did not show any significant difference from controls (Fig. 3.2C, D).
Behavior (mouth hook and body wall contractions)
Simple feeding (mouth hook movements) and a locomotor (body wall
movements) behavior were tested for p-CPA (50mM) and MDMA (10uM) fed larvae.
Larvae ate these compounds from 1st to 3rd instar stage and at the mid 3rd instar
stage they were used for behavioral analysis. Each larva was first assayed for body
wall contractions and then followed for quantifying mouth hook contractions.
Compared to controls p-CPA and MDMA fed larvae showed significant lower body
wall and mouth hook contractions (N=20, Fig. 3.3, ANOVA, P<0.0001). Some of p-
CPA larvae were seen to be crawling with difficulty and some just contracted their
body once or twice.
Spontaneous activity (5-HT and MDMA)
50
The intrinsic activity of the CNS was assessed by monitoring motor
commands to muscles m6 or m7. Both of these muscles receive the same
innervation by two motor axons (Kurdyak et al., 1994). We noted substantial
variation among preparations in the extent of the spontaneous activity and bursting
frequency, but by pulling the edges of the cuticle taut in the experimental chamber
the spontaneous activity can be insured to occur. Measures prior and during
exposure to various compounds gives an approach to determine if the CNS circuit
that initiates the motor commands is sensitive to the particular agent of interest. We
utilized this experimental approach for examining the CNS sensitivity to 5-HT and
MDMA at various doses. However, the approach has some compounding difficulties
in that the preparation can show an enhanced response upon initial exposure but
with rapid desensitizing after only a few minutes depending on the dose and
compound. For example, application of MDMA resulted in a marked busting
behavior followed by short high frequency bursts (Fig. 3.4A for saline and 3.4B for
MDMA). This was subsequently followed by decreased basal activity and inactivity to
evoke a sensory-CNS-motor response by electrical stimulation of an afferent nerve
root. The bursts over time became less frequent and shorter in duration (Fig. 3.4C).
In quantifying the bursting behavior I counted all the peaks within each burst
and recorded the time of each burst. I then counted the frequency of burst per
second and averaged all frequencies. A five second period was taken and a mean
value per second was calculated. The mean frequency obtained before and after
application of drug was used to calculate a percent difference (Fig. 3.5).
51
Spontaneous activity measured in saline controls showed a small increase of
20% in the mean (Fig. 3.5A) but this was not significant. In presence of 5-HT, control
larvae showed a larger dose dependent increase in mean activity (Fig. 3.5A),
whereas a lower concentration of MDMA, 100nM and 10µM, showed a smaller
increase, however MDMA at 100µM showed a decrease in mean activity, but this
was not significant (Fig. 3.5A). There was a substantial amount of variation from
preparation to preparation.
Larvae fed 50mM p-CPA showed a slight increase in activity on application of
10nM and 100nM 5-HT whereas at 10µM a slight decrease in the mean response is
seen but this was not significant (Fig. 3.5B). Probably at this high concentration of 5-
HT the preparations are desensitized. MDMA (100µM) fed larvae showed an
increase in neural activity at all concentrations of 5-HT, although it is a very small
increase (Fig. 3.5C). Even in these preparations a lot of variation was observed. No
statistical significance was detected among any of the groups examined with an
ANOVA P> 0.05 as well also with a non-parametric rank-sum Wilcoxin test.
Sensory-CNS-motor circuit (5-HT and MDMA)
The sensitivity of central circuits to 5-HT and MDMA of various doses was
examined by stimulating sensory nerves and monitoring motor units before and
during exposure to the compounds. There is a clear dose-dependent effect to both
5-HT and MDMA, however in opposing actions, in altering the evoked CNS activity
(Fig. 3.6A). Sham controls were performed since the disturbance of changing
solutions could result in sensory activity and thus drive a motor response. In a few
52
preparations a slight increase occurred but the changes are non-significant for the
group. The preparations were used once for each condition. The mean percent
increase in response to 5-HT as well as the variability from 10 nM, 100 nM and
10�M (ANOVA P<0.0001, n=7). Tukey post hoc test showed only 10�M 5-HT
exposure as significantly different from sham (P<0.05). To our surprise, MDMA
caused a decrease in evoked responsiveness which was dose-dependent except
the variation among preparations was very consistent unlike that for 5-HT at higher
doses (Fig. 3.6A). The MDMA effects are significantly different from shams. (ANOVA
P<0.0001).
p-CPA fed larvae when exposed to 5-HT showed a decreased response. The
decrease in activity is dose dependent. 5-HT at 10µM showed an approximately
50% decrease compared to saline whereas 100nM showed a 30% decrease in
activity (Fig. 3.6B). This was unexpected. Maybe preparations are desensitized very
fast on application of 5-HT. On the other hand, MDMA fed larvae showed an
increase in activity when exposed to 5-HT. Exposure to 10nM 5-HT showed a larger
increase as compared to 100nM or 10µM 5-HT (Fig. 3.6B). But as compared to
shams both p-CPA fed and MDMA fed larvae showed a significant difference
(ANOVA P<0.0001). Also MDMA fed larvae exposed to 10nM 5-HT showed a
significant difference (P<0.05; Tukey test).
Heart Rate
5-HT is known to have a role in cardiac function in Drosophila (Dasari and
Cooper, 2006). So we examined if larvae altered their 5-HT sensitivity in cardiac
53
function to long-term depletion of 5-HT, the larvae were fed p-CPA from 1st to 3rd
instar. In mid-3rd instar the larvae were dissected and heart rate monitored for
alteration to exogenous application of 5-HT. Larval heart rate (HR) increases upon
exposure to 5-HT (100nM); however, there is no significant increase above controls
for larvae depleted of 5-HT (Fig. 3.7). Controls increased HR by 31% and p-CPA fed
larvae by 28%. The significance to examine the HR is that it serves as another
physiological measure, out side the CNS, for the sensitivity to 5-HT in these
pharmacologically manipulated larva.
Levels of Serotonin and Dopamine
It is important to determine the amount of depletion of 5-HT in the CNS as
well as whole body to correlate with the delayed development in p-CPA and MDMA
treated animals. The brains from mid 3rd instar larvae were individually dissected out
of the larvae for HPLC analysis. At least 5 samples with each sample containing 25
pooled brains were analyzed. The HPLC results showed that control brains (n=6)
have about 12-14 pg/brain of 5-HT and DA (Fig. 3.8A). Treatment with p-CPA
(50mM) caused a significant decrease in 5-HT levels in larval brains, approximately
by 90% (n=5, ANOVA, P<0.05 Fig. 3.8A). Larvae that ate p-CPA also resulted in a
significant decrease in DA levels (n=5, AVONA P<0.05, Fig. 3.8A). MDMA
treatments did not produce a difference in the levels of DA or 5-HT (n=5, Fig. 3.8A).
Treatment with 5,7-DHT, a compound that was perdicted to kill serotonergic
neurons, showed a small decrease in the mean 5-HT levels but it was not significant
(Fig. 3.8A).
54
Intact larvae were also collected from the same culture vials as above for
each group and washed in water to remove food residues. 20 larvae were pooled
together for each set and subjected to HPLC. Control larvae had approximately 500
pg/larva of DA and 300pg/larva of 5-HT. No significant differences in levels of 5-HT
and DA occurred with either p-CPA or MDMA treated groups as compared to
controls (Fig. 3.8B).
Larvae were fed different concentrations of p-CPA (0.5, 5, and 50 mM) and
MDMA (10uM, 100uM and 1mM) and allowed to form pupa. Every 4 hrs vials were
checked for newly eclosed adults. The adults were collected and frozen immediately.
The heads were chopped off from these adult flies and 5 heads were pooled for
each set. The analysis of HPLC data revealed no significant difference in DA or 5-
HT levels in either of the groups as compared to controls except for the 1mM MDMA
treated group (Fig. 3.8C, D). The larvae fed 1mM MDMA showed a significant
increase above controls in levels of both 5-HT and DA (n=5, ANOVA, P< 0.05, Fig.
3.8C, D).
DISCUSSION
In this study I demonstrated that feeding larvae p-CPA or MDMA will retard
development and decreases activity associated with crawling and eating
behaviors. HPLC analysis of 5-HT and DA for the whole larvae did not show
any effects of the drug treatments, where as selectively measuring the
brains of larvae showed significant effects. The levels of p-CPA that slowed
development also reduce the concentrations of 5-HT and DA in 3rd instar
larval brains. This effect of p-CPA is dose-dependent. However, treatment
55
with MDMA did not show any alterations in the 5-HT and DA in the CNS of
larvae but in newly eclosed adults, which ate MDMA as larvae, showed a
dose-dependent increase in both 5-HT and DA. Initial exposure of the larval
brain to MDMA caused an increase in spontaneous activity which was short
lived (~3 minutes) and in fact decreased evoked sensory-CNS-motor activity
afterwards. In contrast, 5-HT increased evoked activity significantly but not
spontaneous activity in a dose-dependent nature. When the larvae were depleted of
5-HT throughout development they did not increase their sensitivity to the excitatory
response of 5-HT but showed a more pronounced inhibitory action of 5-HT on the
central circuit. Just the opposite effect occurred for larvae fed MDMA, in that
treatment with MDMA throughout larval development resulted in the central circuit to
be more responsive to 5-HT. The sensitivity of 5-HT on the larval heart was used as
an additional measure of the effects of p-CPA treatment. There was no effect on
sensitivity of heart to 5-HT in p-CPA fed larvae.
Since development in larvae fed p-CPA or MDMA showed a dose-dependent
effect in slowing down the rate of development as well as increasing the mortality
rate, it would appear the involvement of the CNS serotonergic system is vital to the
health of larvae. Direct and indirect effects are both likely to contribute to the offset
development. Since larvae fed p-CPA or MDMA showed reduced mouth hook
movements one would logically ask, "Was a reduced diet a limiting factor?" We
could assay the total amount of food within the digestive system of larvae with a dye
tainted food technique but such approaches do not inform one if the nutrients are
absorbed from the digestive system. Possibly animals absorbed even more nutrients
56
if retention within the digestive system was enhanced due to a decrease gut motility
from treatments with p-CPA or MDMA. Whole animal HPLC assays did not show a
significant decrease in 5-HT with p-CPA treatments and given that 5-HT can
modulated gut motility in many invertebrates (Ayali and Harris-Warrick, 1999; Katz
and Harris-Warrick, 1989) possible there was no effect on digestive processes. But
just as likely there may well be neural regulation of gut motility in larval Drosophila
which is consequentially altered by the decrease in 5-HT within the CNS. Autonomic
neural function of digestive properties in insects (Copenhaver and Taghert, 1989,
1991; Penzlin, 1985; Zavarzin, 1941) and crustaceans (Shuranova et al., 2006) is
established but how the autonomic digestive function maybe regulated by
serotonergic circuits within the CNS has not been addressed.
The development of some circuits within the CNS of arthropods are known to
be dependent on 5-HT. The olfactory neurons in Manduca sexta (moth) are 5-HT
sensitive (Kloppenburg and Hildebrand, 1995) and the correct development of
neuronal processes within the olfactory center is dependent on 5-HT (Hill et al.,
2003). The central development of the olfaction circuits in crustaceans (Sandeman
et al. 1995) and C. elegans (Nuttley et al., 2002) are even dependent on 5-HT. In the
eyes of flies (Chen et al., 1999), as well as crustaceans (Aréchiga and Huberman,
1980), 5-HT alters the visual sensitivity. This in turn could readily have a substantial
effect in development of the neural circuit as it is well established from invertebrates
(Payne, 1911; Roach and Wiersma 1974; Scott et al., 2003; Cooper et al., 2001) to
mammals (Hubel and Wiesel, 1970) that activity in visual system sculpts the
peripheral and central circuits. Since neuroendocrine axis in invertebrates are
57
sensitive to 5-HT (Lee et al., 2000) this could alter development of the whole animal
as well as specific neuronal processes and neurohormones with very broad actions
(Nässel, 2002). Likewise, if the circadian patterns were altered due to decreased
neuronal 5-HT or direct action of MDMA on 5-HT receptors then one would could
expect endocrine related developmental abnormalities.
Genes that regulate tryptophan hydroxylase for the biochemical synthesis of
5-HT are known in Drosophila and there are two different genes encoding for two
different forms of the enzyme. One that is expressed in the periphery and one in the
CNS (Coleman and Neckameyer, 2005). When there are defects in phenylalanine
hydroxylase in mammals a disease state of phenylketonuria can occur (Lenke and
Levy, 1980). Phenylalanine is precursor for tyrosine. In this pathological state it is
known that tyrosine hydroxylase and TPH are inhibited which leads to reduced 5-HT
and associated neuronal damage (Curtius et al., 1981; Roux et al., 1995) which
impinged on developmental rates, thus a similar situation may occur in Drosophila.
The associated higher death rate with larvae fed MDMA and p-CPA in high doses
points to some significance in the serotonergic system for overall health since it is
likely the primary site of drug action.
The slowed developmental time from egg to pupation and pupation to
eclosion with p-CPA and MDMA treatments is surprising since MDMA did not result
in a reduced 5-HT in the larval brain. Thus, the retardation in development cannot be
solely due to reduced 5-HT, but may likely be due to altered central neural activity
since exposure to p-CPA or MDMA changed spontaneous as well as evoked
patterns of central circuits. An easy approach to index rate of development was a
58
time measure for 50% of the larva to pupate or pupa to eclose but this does not
provide a full spectrum of the developmental dynamics, thus we took the laborious
task of checking fly cultures each 4 hrs throughout the day and night for weeks to
obtain the developmental curves. Likewise analysis is best approached to span the
entire curve instead of a single time stamp.
The behavioral similarities, as with overall development, associated with both
p-CPA and MDMA indicates that the overall health of the animal might be comprised
by treatments in a dose-dependent manner. Despite 5-HT levels not declining with
MDMA treatments possible just the dis-synchronization of appropriate neural activity
did not allow the animal to perform coordinated motor commands. Where as the
depletion of 5-HT in the CNS by p-CPA must have produced altered neural activity
by a different means from that of MDMA. 5-HT is associated with modulation of
eating/digestion in crustaceans (Shuranova et al., 2006) and humans (Aubert et al.,
2000) as well as in motor unit coordination (LeBeau et al., 2005; Dasari and
Cooper, 2004; Strawn et al., 2000; Weiger, 1997) and behavior (Bicker 1999; Toth et
al., 2005; Barnes and Sharp, 1999) in a wide variety of animals. Likewise MDMA in
humans promotes mastication and heightened activity for motor function. The few
minutes of increased central motor commands, in the larvae to exposure of MDMA,
prior to the decrease evoked responses is contrary to what was expected since we
predicted that a further increase in synaptic 5-HT release would mimic the
exogenous application of 5-HT. However the proposed synaptic model of MDMA
action in mammals (Green et al., 2003; Simantov, 2004; Sprague and Nichols, 2005)
may not hold for Drosophila. In addition, the surprising results of p-CPA reducing not
59
only 5-HT but also DA beacons careful assessment of extrapolating mechanisms of
drug action noted in mammals to invertebrates. Such oversights have also been
noted to occur for pharmacological serotonergic agents used in crustacean
behavioral research (Sparks et al., 2003) which is due to various cellular cascades
and receptor subtypes (Clark et al., 2004; Tierney, 2001).
5-HT and MDMA showed an increase in spontaneous activity. p-CPA and
MDMA fed larvae also showed an increase in presence of 5-HT. An opposite effect
was seen with spontaneous activity. Sensory-CNS-motor circuit is modulated by 5-
HT (10µM) in a biphasic manner (Dasari and Cooper, 2004). Here I have shown that
5-HT alters evoked activity in a dose dependent manner. In Tritonia, increased
levels of 5-HT enhances the abilty of dorsal swim neurons to initiate rhythmic activity
in swim motor neurons. (Fickbohm et al., 2000). To my surprise the firing frequency
for motor neurons in p-CPA fed larvae reduced in presence exogenous 5-HT. Since
5-HT levels are reduced throughout development, the receptors are not expressed
properly. But opposite effects are seen with MDMA. Application of MDMA decreased
the firing frequency but MDMA fed larvae showed an increase in activity with 5-HT
application. As mentioned earlier, MDMA may not be completely working through the
5-HT system. In humans one of the mechanisms by which MDMA works is by
reversing 5-HT transporter. Drosophila 5-HT transporter (dSERT) is homologous to
human and rat SERT (51%). Demchyshyn et al. (1994) showed that dSERT is not
similar to hSERT pharmacologically. The affinity of dSERT is different for
antidepressants like imaprimine. MDMA may not be acting on dSERT in the same
manner as for hSERT.
60
I was fortunate to have carried out the HPLC analysis, as I would not have
realized the broad action of p-CPA in reducing dopamine in parallel with serotonin. It
appears that p-CPA likely works on tryptophan hydroxylase (TPH) as well as the
biochemical pathway for synthesis of DA in Drosophila. The likely enzyme targeted
for by p-CPA in DA production is tryptophan-phenylalanine hydroxylase. This
enzyme is also used in part for 5-HT production for Drosophila (Coleman and
Neckameyer, 2005). The long-term treatment of larva that were followed throughout
pupa and eclosin offered even more fodder for speculations since the actions were
dose-dependent in increasing 5-HT as well as DA in adults when treated with
MDMA. The p-CPA decreased both 5-HT and DA in new adults much the same as
was observed for larva. The speculation at present is that MDMA promotes
production of 5-HT and DA by either turning off inhibitory feedback balance or
directly stimulating biosynthesis in the CNS. We also noted that levels of 5-HT or
DA in the CNS do not parallel whole larval body analysis and p-CPA treatments had
no discernable effect on the whole body 5-HT levels. Tissue specific analysis is
required in this model organism for 5-HT and DA. It is possible that p-CPA may only
work on the TPH enzyme produced centrally since it is coded by a different gene
than for the one expressed peripherally (Coleman and Neckameyer, 2005). As in
vertebrates there are differential expressed TPH genes centrally and peripherally
which produce different enzyme isoforms (Walther et al., 2003). It is possible even
if the gene sequence is the same they may undergo alternative splicing differentially
making one form more sensitive to drug treatments. Enzyme splicing variants which
have various drug affinities as well as various 5-HT receptor variants and affinities
61
are now commonly observed in multiple species (Kishore and Stamm, 2006; Krobert
and Levy, 2002). With the dual effects of p-CPA on 5-HT and DA concentrations in
the CNS, one now has to determine if the observed behavioral changes are related
to alterations in 5-HT or DA as well as the delayed development. As in vertebrates,
DA in Drosophila has an effect in behavior and locomotion (Kume et al., 2005;
Cooper and Neckameyer, 1999; Neckameyer, 1996, 1998).
The larval heart did not show any altered responsiveness to exogenously
applied 5-HT (10uM) in the animals fed p-CPA most likely since peripheral 5-HT
levels were not altered. Since peripheral 5-HT levels were not altered as determined
by HPLC we did not expect a change. The heart does serve as a independent assay
for alteration in the CNS responses to 5-HT since the heart is also responsive to 5-
HT (Dasari and Cooper, 2006); however, the aorta of the larval heart does appear to
be innervated (Johnstone and Cooper, 2006) and neuromodulatory action on this
innervation is not known. This is one reason in my studies that the CNS was
removed when conducting the heart assay.
These studies have produced some unexpected findings with MDMA's
actions, particularly that 5-HT was not depleted in the brains of larvae and raised in
pupa/adults for larvae fed MDMA. Possible enzymatic assays would resolve if
MDMA could stimulate synthesis. The pronounced inhibitory effects of 5-HT after p-
CPA treatment also was surprising since acute application produced excitation of the
central circuit. In addition, the reduction of dopamine by p-CPA treatment in larvae
was serendipitously found which could be accounted for by p-CPA not only blocking
TPH but also tryptophan phenylalanine hydroxylase and tryosine hydroxylase that is
62
used to produce dopamine. To resolve this issue enzymatic analysis is needed. The
excitatory and depressing effects of 5-HT could be accounted for by alternative
regulation of the four known 5-HT receptor subtypes or even alternative splicing of
the D5-HT2 subtype as is known to happen in mammals (Kishore and Stamm, 2006;
Pauwels, 2000). Since out of the four (5-HT7Dro, 5-HT1ADro, 5-HT1BDro, and 5-HT2Dro)
receptor subtypes, that are analogous to mammalian systems in classification, a
possible up regulation of the 5-HT1 subtypes could cause a depression of cellular
excitability (Barnes and Sharp, 1999; Tierney, 2001; Nichols et al., 2002). Where in
a neuronal circuit the locations of the particular receptors subtypes could result in
excitation on one set that ultimately inhibits motor neurons through a GABA-ergic
path. Obviously more work is needed to know receptor localizations, expression
regulation and cellular responses to make sense of the full sensory-CNS-motor
circuits described in this study as well as in studies on vertebrates neuronal circuits
(McMahon et al., 2001; Sodhi and Sanders-Bush, 2004; Vitalis and Parnavelas,
2003). With more experimentation, one may understand the mechanistic cellular
actions of various pharmacological agents and the potential role of endogenous
neuromodulators on the serotonin-ergic circuits and developmental influences on the
larval brain as well as the brain during the pupa to adult transformation.
63
FIGURES:
Figure 3.1: p-PCA growth curve:
p-CPA was fed from 1st instar to 3rd instar stage. The time to pupation from eggs and
time to eclosion from pupation is calculated for each larva. A) Cumulative sum for
time to pupation from eggs at different concentrations of p-CPA. B) Relative
cumulative sum for A. Larva fed highest concentration of p-CPA (50mM) took the
longest time to pupation. 50% of larva took 100 hrs longer to pupate as compared to
control and lower concentration of p-CPA. C) Cumulative sum for time to eclosion
from pupa formation for different concentrations of p-CPA. D) Relative cumulative
sum for C. Time for each pupa is adjusted to ‘0’ and calculated time taken from
pupation to eclosion. The larva fed 50mM pupa took longest time to eclose but they
seem to catch up with the others in the eclosion.
64
Figure 3.2: MDMA growth Curve.
MDMA was fed from 1st instar to 3rd instar stage. The time to pupation from eggs and
time to eclosion from pupation is calculated for each larva.
A) Cumulative sum for time to pupation from eggs at different concentrations of
MDMA. B) Relative cumulative sum for A. 50% of larvae took ~ 165 hrs for pupation
at the highest concentration of MDMA, 1mM.
C) Cumulative sum for time to eclosion from pupa formation for different
concentrations of MDMA. D) Relative cumulative sum for C. As described earlier,
time for each pupa is adjusted to ‘0’ and calculated time taken from pupation to
eclosion. Pupa of 1mM MDMA fed larvae took a little longer (~ 5 hrs more than
controls) to eclose as compared to lower concentrations.
65
Figure 3.3: Body wall and mouth hook contractions.
A) Body wall contractions were counted for one minute and averaged per minute. p-
CPA or MDMA fed larvae showed a decrease in body wall movements as compared
to controls (N =20, ANOVA<0.05). B) Mouth hook contractions were counted in
yeast solution for one minute and averaged per minute. Both p-CPA and MDMA fed
larvae showed a decrease in mouth hook movements as compared to controls (N
=20, ANOVA<0.05)
66
Figure 3.4: Spontaneous activity in 3rd instar CS larvae.
A) Spontaneous activity in saline. B) Spontaneous activity in presence of 10�M
MDMA. C) Total recording of spontaneous activity in presence of saline and MDMA
10�M. Activity in presence of MDMA initially increased for first few 100s of seconds
and decreased thereafter.
67
Figure 3.5: Spontaneous activity.
The % change in average frequency per second (activity) from saline in CS larvae
(A), p-CPA fed larvae (B) and MDMA fed larvae (C). 3 different concentrations of 5-
HT and MDMA are used on CS larvae and for the p-CPA and MDMA fed larvae 3
different concentrations of 5-HT are used in comparison with saline. The number
above the bar shows the sample size.
68
Figure 3.6: Sensory-CNS-motor circuit.
A) A percent difference in the activity of motor units to muscle 6 before and after
application of 5-HT or MDMA. Saline shams showed a small increase. 5-HT showed
a dose depended significant increase, whereas MDMA showed a dose depended
significant decrease in activity from shams (ANOVA, P<0.0001).
B) 50mM p-CPA fed larvae and 100µM MDMA fed larvae were exposed to 5-HT of
different concentrations. The percent difference in activity is recorded before and
after 5-HT exposure. p-CPA fed larvae showed a significant decrease in sensitivity
to 5-HT whereas MDMA fed larvae showed a significant increase in activity in
presence of 5-HT. (ANOVA, p<0.0001).
69
Figure 3.7: Heart rate.
The percent change in heart rate upon exposure to 5-HT (100 nM). Both control and
larvae fed p-CPA showed a similar and significant effect (n=7; P<0.05, Student's t-
test) increase in heart rate without a significant differences between the groups.
70
Figure 3.8: HPLC analysis of 3rd instar larvae.
(A) HPLC analysis on 3rd instar larval brains. P-CPA treated larval brains (n=5)
showed a decrease in DA levels (open bar, ANOVA <0.05) and 5-HT levels (hashed
bars, ANOVA, P<0.05). MDMA and 5,7-DHT didn’t show a difference (n=5 and 3
respectively). (B) DA and 5-HT levels in whole larvae. None of treated groups
showed any significant difference from the control group. (n=5). (C) DA levels in
adult heads, collected after they have been treated with drugs from 1st to 3rd instar
stage. 1mM MDMA showed a significant increase in DA levels (n=5, ANOVA,
P<0.05). (D) 5-HT levels in adult heads. Except for 1mM MDMA, the other groups
did not show any difference in levels compared to controls. (n=5).
71
CHAPTER 4
KNOCK DOWN OF 5-HT2 RECEPTORS ALTERS DEVELOPMENT, BEHAVIOR
AND CNS ACTIVITY IN DROSOPHILA MELANOGASTER
ABSTRACT
The expression of anti-sense 5-HT2dro receptor retards larval development
and produces slower body movements and gustation. When expressed from 1st
instar, induced by heat shock, a high of degree of death occurred and few reached
3rd instar. The CNS of larva increases its excitability when exposed to 5-HT, however
when the 5-HT2dro receptor is reduced in expression the sensitivity to exogenously
applied 5-HT was decreased. Evoked sensory-CNS-motor circuits as well as
spontaneous motor neuronal activity is reduced in larvae in which the 5-HT2dro
receptor is knocked down. Like CNS activity, heart rate (HR) in larva is sensitive to
5-HT. The knock down of 5-HT2dro receptors from 1st instar to early 3rd instar resulted
in no effect to sensitivity to HR although the initial HR was lower. Thus, the 5-HT2dro
receptor required for normal body development and CNS responsiveness to 5-HT.
INTRODUCTION
Serotonin (5-HT) is a major neurotransmitter and neuromodulator in both
vertebrates and invertebrates. It has been shown to have a role in development and
behaviors of various vertebrates and insects (Whitaker-Azmitia, 2001; Lucki, 1998;
Monastirioti, 1999; Osborne, 1996; Chapter 3 for this dissertation). Recently, it was
shown that 5-HT increases activity in a sensory-CNS-motor circuit in larvae (Dasari
and Cooper, 2004). When reducing the production of 5-HT, by pharmacological
72
means through feeding p-CPA to larva, there is a delay in the rate of development
and slowed body wall as well as eating behavior (Chapter 3 for this dissertation;
Dasari et al., 2006). Likewise, exposure to MDMA (ecstasy), a drug of abuse, which
impacts on the serotonergic system, also results in slowed larval development and
reduced bodily movements (Dasari et al., 2006). Thus, altering the production or the
level of 5-HT has broad ranged effects in larval Drosophila, however to understand
the mechanisms by which these alterations occur one needs to address the effectors
of 5-HT which relay the signals to cells.
5-HT acts through multiple receptors to mediate its many functions in various
tissues. There are 14 known 5-HT receptors in vertebrates (Barnes and Sharp,
1999) whereas four are known to occur in the Drosophila genome (Tierney, 2001).
Hence Drosophila melanogaster forms an attractive model organism to study the
role of 5-HT mediated by these receptors. The four known receptors are named as
5-HT7do, 5-HT1Adro, 5-HT1Bdro, and 5-HT2dro (Witz, 1990; Saudou et al, 1992; Colas et
al, 1994; Tierney, 2001).
The 5-HT2dro receptor subtype is of particular interest since in humans 5-HT2
receptor is known to be associated with many diseases such as schizophrenia,
depression, and anxiety (Leonard 1994; Fuller 1991). In addition, some drugs of
abuse work on the 5-HT2 receptor subtypes to relay the effects sort after by addicts
(i.e., lysergic acid dimeythlamide) (Roth et al., 1998). Drosophila offer some
advantages in the ability to address the developmental and mechanistic
understanding of neuromodulators, particular 5-HT since this model organisms is
73
being increasing used to address physiological related inquiry as well as the genetic
understanding of diseases that inflict humans (Kendler and Greenspan, 2006).
Colas et al. (1999a,b) showed that 5-HT2dro is essential during embryo-
genesis and that it is the major receptor subtype in 3rd instar larvae (Colas et al.,
1994). One approach to determine the role of 5-HT2dro is to selectively knock down
its expression. In my investigation I use two temperature sensitive transgenic lines,
HsZ2 and Y32 which were designed to specifically reduce the functional expression
of the 5-HT2dro receptor. HsZ2 strain is a 5-HT2dro anti-sense under heat shock
GAL4-UAS construct. The Y32 strain expresses a 5-HT2dro anti-sense under a heat
shock promoter. Since these two strains had not been fully characterized for
temperature induction of the anti-sense and the role on development, I conducted
studies at 18, 21 and 33 °C. To determine the effects of reduced 5-HT2dro expression
on behavior and on CNS receptivity to 5-HT studies at low and heat shock induced
temperatures were compared for sham controls and the two strains.
Johnson et al., (1997) have shown heart rate to be modulated by 5-HT in
Drosophila. Also recently we have shown direct effects of various doses of 5-HT on
Drosophila heart (Dasari and Cooper, 2006). 5-HT2B receptor in mammalian model
organisms have been shown to regulate cardiac embryonic development and
cardiac adult functions. Hence these receptors might be involved in cardiac
pathophysiology in adults. We measured heart rate in transgenic lines to investigate
if 5-HT2dro receptor regulates it.
74
METHODS
Stock and Staging of Larve
The common ‘wild-type’ laboratory strain of Drosophila melanogaster, Canton
S, was used in these studies as controls. HsZ2 and Y32 are used as experimental
lines. HsZ2 is a 5-HT2dro antisense under GAL4-UAS system and Y32 is a 5-HT2dro
antisense under heat shock promoter (gifts from Dr. L. Maroteaux, IGBMC-CNRS-
INSERM, Universite de Strasbourg, France). The methods used to stage fly larvae
have been described previously (Campos-Ortega and Hartenstein, 1985; Li and
Cooper, 2001). All animals were maintained in vials partially filled with a cornmeal-
agar-dextrose-yeast medium. All animals were kept on a 12:12 light-dark cycle.
Behavioral Assays
Early 3rd instar larvae were used for behavioral assays. Feeding and
locomotor behaviors were assessed as described in Neckameyer (1996) and Li et
al., (2001). In brief, single animals were placed on a 2% agar surface and the
number of body wall contractions was counted for 1 minute, after which an animal
was placed in a 2% yeast solution overlaid on an agar plate (just covering the larvae
with allowing the spiracles to reach out of the solution). In this condition, Drosophila
larvae immediately feed, initiating a pattern of repetitive mouth hook movements.
The number of full mouth hook contractions in 1 minute was counted (Sewell et al.
1975).
Dissection and electrophysiological recordings
75
Wandering 3rd instar larvae were dissected as described earlier (Cooper et
al., 1995). In brief larvae were dissected ventrally removing heart and viscera which
left a filleted larvae containing only a body wall, body wall muscles and the neural
circuitry for the sensory, CNS and body wall (i.e., skeletal) motor units. The HL3
saline was prepared in the lab from component reagents (Sigma) and contained:
1.0 mM CaCl2·2H2O, 20 MgCl2, 70 mM NaCl, 5 mM KCl, 10 mM NaHCO3, 5 mM
trehalose, 115 mM sucrose, and 5 mM BES (N,N-bis[2-Hydoxyethyl]-2-
aminoethanesulfonic acid) (Stewart et al., 1994).
The recording arrangement was essentially the same as previously described
(Neckameyer and Cooper, 1998; Stewart et al., 1994). Intracellular recordings in
muscles were made with 3 M KCl-filled microelectrodes (30–60 M ). The amplitudes
of the excitatory postsynaptic potentials (EPSP) elicited by Is and Ib motor nerve
terminals in the various segments of muscles m6 were monitored. Intracellular
responses were recorded with a 1×LU head stage and an Axoclamp 2A amplifier.
Sensory-CNS-Motor circuit
To induce a sensory-CNS-motor circuit the tail segmental nerves were cut
and stimulated using the suction electrode (Dasari and Cooper 2004). The stimulator
(S-88, Grass) output was passed through a stimulus isolation unit in order to alter
polarity and gain (SIU5, Grass). Stimuli were given in short bursts at a frequency of
40Hz (10 stimuli at 40Hz). The amount of voltage given was adjusted so as to recruit
sensory axons to produce at least a few responses in the muscles so that if any
further increase or decrease occurred due to the effects of a neuromodulator the
76
change could be obtained. This sensory stimulation leads to interneurons and motor
neurons to be activated, thus inducing a response in muscle 6.
In cases where the intrinsic spontaneous activity was measured all the
segmental nerves were left intact. The intrinsic CNS activity probably is induced by
the fact the animal has been cut down the dorsal midline and stretched out on a dish
by pinning four corners of the animal down. The burst frequency and frequency
within a burst were measured for experimental comparisons. All experiments were
performed at room temperature (20–22 °C).
Heart Rate (HR) measures
The same microscopic method as for behavioral movements was used to
record HR but with the exception of a 2x base objective to obtain a higher resolution
of the heart and trachea. Early 3rd instar larvae were dissected ventrally and pinned
on 4 corners. The heart and trachea are exposed in these semi-intact preparations.
The movements of the trachea or heart were used for direct counts (Dasari and
Cooper, 2006). Effects of 5-HT were observed in CS, HsZ2 and Y32 larvae allowed
to grow at different temperatures.
Developmental assays Eggs were collected after a 15-minute prepulse for 2 hrs and allowed to
develop at 21oC until hatching. At which time 3 different developmental temperatures
were investigated for the effects on development and I tested if room temperature
might be a temperature at which a low expression level of the heat shock promotor
77
might be active which could result in anti-sense production in the two experimental
strains.
1st instars (about 15) were transferred to vials containing food and placed at
given temperature. The food was made with standard corn meal diet (Neckameyer,
1996; Li and Cooper, 2001). When wandering 3rds were seen, the vials were
checked every 4 hrs and each individual pupa was marked on the walls of the tube
to recorded the time to pupation. This was done throughout the day and night. The
time to pupation and the time spent as a pupa, until eclosin, were indices for
development.
RESULTS
Behavior – Mouth hook and body wall movements
Locomotion (body wall) and feeding (mouth hook) movements were
measured for CS, HSZ2 and Y32 strains at permissive (room temperature) and
restrictive temperatures to induce the heat shock antisense production of the 5-
HT2dro receptor. There was no significant difference seen among the three lines in
either body wall or the mouth hook movements when larvae were raised in room
temperature (ANOVA, N > 10, Fig. 4.1). To activate 5-HT2dro antisense production,
early 3rd instars were given a 4 hr heat pulse at 33oC and then the locomotor and
feeding behaviors were measured at room temperature within 5 minutes of taking
them out of 33oC. Each larva was taken from the incubator and placed on a test
plate. This allowed the larvae to become familiar with the test plate for a minute.
Then body wall and mouth hook movements were measured. HsZ2 line showed
78
lower average body wall movement than CS (Fig. 4.2 ANOVA, P<0.01, N = 25). Y32
also showed lower body wall movements than CS (Fig. 4.2 ANOVA, P<0.05, N=25).
But no significant difference was seen in mouth hook movements among the 3 lines
that were heat shocked (Fig. 4.2, N = 25).
To control for the temperature variations I measured these feeding and
locomotor behaviors at 33oC after a 4hr heat pulse at the same temperature. Here
again no significant difference in the mouth hook movements occurred (Fig. 4.3B,
N=10) but this time there was no significant difference for body wall movements
(Fig. 4.3A, N=10).
To see the effects of a reduced expression of 5-HT2dro throughout the
development stages, I grew larvae from 1st instar to 3rd instar at 31-32oC. Early 3rd
instars were used for measuring body wall and mouth hook movements at room
temperature. To my surprise there was no significant difference between HSZ2 and
Y32 compared to CS (Fig. 4.4A, B, N > 10).
Larval Development Developmental assay were carried at out at 18oC, room temperature (21-
22oC) and 31-32oC for CS, HsZ2 and Y32. Time was assessed for 50% of
population to reach pupation or eclosion. The time eggs were collected is taken as a
zero point. At low temperature, where the anti-sense for 5-HT2dro is not being
expressed, we did not anticipate to see any major difference in the development
patterns. But a small variation was observed between the 3 groups for time to
pupation (Fig. 4.5A, B). 50% of the population in the CS group took approximately
79
244 hrs for pupation, whereas for Y32 50% took 280 hrs and 50% of HsZ2 took ~ 8
hrs more than the CS group. This small variation continued for the time from
pupation to eclosion. For calculating the amount of time taken for eclosion for each
pupa, the time point at which pupa formed was set to ‘0’ (zero) and number of hrs
are calculated to eclosion. 50% of pupa for CS and HsZ2 took ~ 190 hrs and ~ 180
hrs respectively, whereas Y32 took ~ 220 hrs (Fig. 4.5C, D). This is a small
difference for CS, HsZ2 and Y32 in both time to pupation and eclosion for 50% of
the population. To statistically test for a difference in the distributions a Kolmogrov-
Smirnov (K-S) test was performed. D and P values for pupation among all groups
were not significant (For CS and Hsz2: D is 0.1507 and a P of 0.985, CS and Y32:
D, is 0.30 and a P of 0.417, HsZ2 and Y32: D, is 0.2549 and a P of 0.610). Similarly
eclosion at room temperature is not significant (CS and HsZ2: D, is 0.0981 and a P
of 1.0, CS and Y32: D, is 0.1593 and a P of 0.908, HsZ2 and Y32: D, is 0.1 and a P
of 1.0)
Similarly, I observed the developmental pattern for room temperature in all 3
groups. Here again I found a small variation in the time taken for 50% of population
to pupate and eclose. 50% of CS and HsZ2 took almost the same amount of time to
pupate (~ 132 and 133 hrs respectively), whereas Y32 took ~ 140hrs (Fig. 4.6A, B).
For eclosion time 50% of HsZ2 took ~100hrs where as Y32 and CS took ~2 and 4
hrs less than HsZ2. A K-S test was used to assess for statistical significant
differences in the distributions. No significant difference was seen for pupation in any
groups (CS and Y32: D, is 0.1442 and a P of 0.996, CS and HsZ2: D, is 0.1462 and
a P of 0.999, HsZ2 and Y32: D, is 0.1625 and a P of 0.993). No significant difference
80
is seen in eclosion time for any groups (CS and HsZ2: D, is 0.2273 and a P of 0.914,
CS and Y32: D, is 0.1697 and a P of 0.986, HsZ2 and Y32: D, is 0.1697 and a P of
0.986).
Developmental effects at the high temperature in which the anti-sense for 5-
HT2dro is actively expressed from 1st instar stage had pronounced effects. 50% of
CS population took 120 hrs to pupate whereas Y32 and HsZ2 took 140 hrs (Fig.
4.7A, B). The major difference observed was when the majority of the CS larvae
reached pupation. Only 3 of 100 HsZ2 and 30 of 100 Y32 had pupated. K-S test
showed a statistical difference in pupation time between CS and HsZ2 (D is 0.5294
with a corresponding P of 0.010) and HsZ2 and Y32 (D is 0.4794 with a
corresponding P of 0.019) but no difference between CS and Y32 (D is 0.3706 with
a corresponding P of 0.123) is present.
Examining the effects on pupation proved to be futile since all of Y32 pupa
and HsZ2 died and 20% of CS pupae eclosed. 50% of CS pupae that did eclose
took 76 hrs (Fig. 4.7A, B).
Spontaneous activity The intrinsic activity was measured from m6 or m7 muscles as both these
muscles receive the same innervation by two motor neurons Is and Ib. The
spontaneous activity is seen when the larva is stretched and pinned, which likely
keeps sensory neurons active. A lot of variation was observed from preparation to
preparation as noted in my earlier studies (Chapters 2-3 of this dissertation). Two
sets of experimental paradigms were used here: (1) larvae were grown at room
81
temperature and neural activity assessed at room temperature; (2) larvae were
grown at 31-32oC and intrinsic activity was measured at room temperature saline.
The sensitivity to 100nM 5-HT was examined for all the groups in these two
conditions.
Intrinsic activity was recorded for 2 min in saline and then another 6-10 min in
the presence of 5-HT. For quantifying, I calculated the average frequency per
second by counting all the peaks within each burst. Also the time for each burst was
recorded. The average frequency per second in saline and in presence of 5-HT was
used to calculate the percent difference in activity from saline.
At room temperature 5-HT (100nM) sensitivity of the CS increased by
approximately 15%, whereas HsZ2 and Y32 showed a decreased responsiveness
(Fig. 4.8A). There is no statistical significance seen among CS, HsZ2 and Y32 (N =
5, ANOVA, Fig. 4.8A). However, the CS and HsZ2 larvae grown at 31-32oC and
exposed to 5-HT (100nM) showed the opposite effects (Fig. 4.8B). CS and HsZ2
showed a decrease response whereas Y32 showed a significant increase in activity
from saline (N = 5, ANOVA, P < 0.013 and Tukey post hoc test, P< 0.05, Fig. 4.8B).
Y32 did not have much activity in saline when compared to that of CS and HsZ2.
Introducing 5-HT increased the spontaneous activity in Y32.
Sensory-CNS-motor circuits
The sensitivity of central circuits to 5-HT in transgenic lines was examined by
stimulating sensory nerves and monitoring motor units before and during exposure
to the compounds. CS showed an increased in motor activity in presence of 5-HT
82
(100nM) at room temperature. Similarly HsZ2 and Y32 showed an increase in
activity at room temperature in response to 5-HT (100nM). No statistical difference
was seen among 3 groups (N = 5, ANOVA, P < 0.15, Fig. 4.9A). When antisense 5-
HT2dro was activated, sensitivity of CNS-circuit to 5-HT (100nM) is changed. CS and
HsZ2 that were grown at high temperature (31-32oC) showed a very small increase
in motor activity. Here again there is lot of variation in preparations. On the other
hand Y32 grown at high temperature showed a decrease in activity. Y32 larvae
grown at 31-32C have a lower activity in saline and there was a fast run down in the
Y32 preparations. The frequency of stimulation had to be increased in some
preparations to 60Hz from the 40Hz, as used for all the other preparations, in order
to evoke motor nerve activity. Again no statistical significance is seen among the 3
groups (N =5, ANOVA, P < 0.23, Fig. 4.9B).
Heart rate 5-HT is known to modulate heart rate in Drosophila larvae (Dasari and
Cooper, 2006) and also recently the larval heart was noted to be innervated
(Johnstone et al., 2006). Hence, I wished to measure the sensitivity of the heart to 5-
HT in transgenic larvae with a reduced expression in the 5-HT2dro receptor to
determine if this receptor subtype had any role. Mid 3rd instars were dissected and
heart rate was monitored. To quantify the responsiveness a percent difference is
calculated for each preparation from saline to 5-HT and an average value is
compared (Dasari and Cooper, 2006). CS, HsZ2 and Y32 larvae grown at room
temperature did not have any significant difference in sensitivity to 5-HT. All the 3
83
groups have shown an increase in the heart rate in presence of 5-HT, though HsZ2
showed a little higher increase, but not statistically significant (N = 10, ANOVA, P <
0.1), than both CS and Y32 (Fig. 4.10A). Larvae that were grown at 31-32oC were
taken out one at a time right before the experiment and heart rate counted at room
temperature for one min in saline and another 3 min in presence of 5-HT. The total
time taken for each preparation is about 5 min. The initial heart rate in saline was
low for all three strains CS, HsZ2 and Y32 as compared to the ones that were raised
at room temperature. CS larvae showed a small variation in its initial heart rate as
compared to that of room temperature. Y32 had a very low HR with some
preparations not beating at all, but when the 5-HT containing saline was introduced
HR increased. When initial heart rates are compared among the three groups that
were raised at high temperature there is a statistically significant difference (Fig.
4.10C) Because of the initial low heart rate, a percent difference was calculated for
comparisons, but in some cases a large difference is then obtained. No statistical
difference is present among the 3 groups (N =10, ANOVA, P< 0.41 Fig. 4.10B).
DISCUSSION
In this study two lines of 5-HT2dro transgenics (Y32 an anti-sense 5-HT2dro
under a heat shock promoter and HsZ2 an anti-sense 5-HT2dro under heat shock
Gal4-UAS promoter) were used to asses effects that would result from the lack of a
5-HT2dro receptor. These transgenics had developmental, behavior and physiological
defects. The development of Y32 and HsZ2 at room temperature and low
temperature are not different from controls. However at high temperature (31-32oC)
84
most of CS formed pupa but just a few Y32 (30%) and HsZ2 (3%) larvae pupated.
Only 20% of CS pupa eclosed and none from the transgenic lines. But no significant
difference is present in simple locomotor movements (body wall and mouth hook
movements) in larvae that are grown at high temperature. However, the
physiological responses of the CNS to 5-HT application in transgenic larvae grown
at high temperature had a decreased responsiveness compared to controls. Though
initial HR was lower in transgenic larvae as compared to that of at room
temperature, no effect on sensitivity to 5-HT was seen.
Studies altering synaptic transmission of adult vertebrate motor nerve
terminals has provided evidence that established synapses and nerve terminals
maintain a high degree of plasticity. A classic example is the increased branching
and growth of motor nerve terminals when the terminal is exposed to botulinum
toxins that block synaptic transmission (Shupliakov et al., 2002). Many of the same
regulating factors in synaptic maintenance in adult animals are likely utilized during
developmental stages, so understanding how synapses develop may have
ramifications in mature synapses. Sokolowski (1980) showed that the behavioral
repertoire of Drosophila larvae requires flexible synaptic inputs. It is likely that
studies conducted at the relatively simplistic NMJ of Drosophila in synaptic
regulation are paralleled in the larval CNS.
The development in the larval Drosophila CNS is a topic that has not been
intensively studied but has been gaining a substantial interest in recent years
(Bossing and Brand, 2002, 2006). Since the larval CNS contains substantially fewer
neurons (~2000, Nassif et al., 2003) as compared to the 100,000 (Iyengar et al.,
85
2006) or more in the adult, the regulatory factors might be easier to assess and
quantify in the larval brain. In addition, since associative learning assays are now
established for larva correlative studies of CNS development or manipulations in
development can be approached in this simpler CNS. It has also been recently
established that the larval CNS is modular in neuropiles (Nassif et al., 2003;
Younossi-Hartenstein et al., 2003) that can be an asset to quantifying alterations in
subdivisions as compared to other regions (Iyengar et al., 2006). Since some of the
regions in the larval CNS are known to be associated with particular sensory
neurons, i.e. olfaction or gustatory senses, then those regions in the CNS can be
examined for behavioral transgenics in olfactory learning screens.
Most of the interest in larval development of synapses has been directed at
understanding factors that regulate NMJ formation and maintenance. The basic
principles that are being uncovered at the NMJ are similar in some regards to the
development within the CNS but with different cues than from muscle. The synaptic
connections at the NMJ formed during embryogenesis are not "hardwired". In target-
removed experiments, Cash et al. (1992) showed that motor neurons could
innervate other muscle fibers when their own targets are missing. These alternate
synapses are physiological functional. However, little or no investigation has been
approached as to the potential effects of altered CNS function form neuromodulators
on the development and maintenance of the NMJs.
The 5-HT2dro of Drosophila is orthologous to mammalian 5-HT2 receptor
subfamily. Colas et al., (1999a,b) reported that 5-HT2dro mRNA expresses during
embryogenesis and gastrulation. With the use of these 5-HT2dro transgenics it has
86
been shown that the receptor must have some functional role in embroygenesis
since ectoderm extension, during embryogenesis, is delayed and hence embryonic
lethality is observed (Colas et al., 1999a,b). In my studies, I activated the heat shock
promoter at 1st instar stage, which retarded the development of larvae and reduced
the number that reached the pupal stage. The ones, which did pupate, however did
not eclose. Like in other animals 5-HT is thought to have a major role during
development. 5-HT is detected in sea-urchins (Buznikov et al., 2005), chicken and
frogs during zygotic divisions in which gastrulation and neurulation takes place.
Nebigil et al., (2000) had shown that mice deficient in 5-HT2B receptor even have a
defective heart.
Since 5-HT modulates locomotive and behaviors in insects and that 5-HT
containing neurons are known to innverate guts, pharyngeal muscles and ring gland
in Drosophila (Valles and White 1988), I assumed that the knock down of the 5-HT2
receptor would produce very pronounced effects on eating behaviors. It was
surprising that no significant effect on locomotor or feeding behavior were obvious.
The rationale for explaining the lack of an effect is probably that these behaviors are
regulated by other 5-HT receptors and not the 5-HT2dro. Recently the 5-HT1 receptor
was shown to have a role in sleep behavior in Drosophila (Yuan et al., 2006). Unlike
Drosophila, the 5-HT2A in mammals is involved in feeding behavior and 5-HT2C in
regulating ones size of meals (Lucki, 1998).
Other neuromodulators that are known to be in the hemolymph and released
into the nuropile of the larval CNS, such as octopamine and dopamine, are likely to
have key roles in shaping the development of the neural circuits (Monastirioti, 1999).
87
Other neuromodulators or peptides may even have a more dramatic role than 5-HT.
Recent work (Pauly et al., 2006) in which larva were feed AMVT (10mg/ml of food),
a drug that blocks tyrosine hydroxylase and thus blocks the synthesis of dopamine,
causes sever developmental problems. If 1st or 2nd instars are fed this compound
they die, however if 3rd instar are fed a majority die as pupa. The ones that do
eclose have white eyes and the cuticle is pale. I have examined dopamine and
octopamine on the CNS circuit of 3rd instar in an earlier study (chapter 2) and
showed that dopamine (10�M) does increase the activity of the circuit, however
octopamine at the same concentration resulted in the preparations to undergo
massive waves of contraction.
Since there is such a pronounced effect of octopamine on the neural activity
one would expect a deficit in the production of the receptors to results in substantial
changes in CNS development. As far as I am aware, no such studies have yet to be
conducted. The effects of octopamine and a related compound, tyramine, have been
examined in relation to honey bee behavior and flight (Fussnecker et al., 2006). In
order to block the action of these compounds antagonists were injected into the
animals. So similar pharmacological approaches could be used in larval Drosophila
however feeding the compounds might be less problematic. One does have to worry
that the compounds mixed in with food and fed to larva or adults might be broken
down during digestion.
It is likely that similar developmental cues such as activity and local
retrograde signaling factors hold for synapse formation and maintenance in the
larval CNS as established in other model preparations, however detailed studies on
88
defined tracts are necessary to substantiate the various possibilities. Possible by
using a fly construct that expressed GFP in a subset of sensory neurons in the Y32
or HSZ fly strains and then imaged within a 3rd instar larval CNS that had the 5-
HT2dro receptor knocked down, the effect of the 5-HT2dro on the development of the
sensory circuits could be addressed. The established techniques of GFP expression
(Brand, 1999), Gal4/UAS (Brand and Perrimon, 1993), and the MARCM (Lee and
Luo, 1999) offer one a powerful approach to examining identified neurons during
development and their reactions to manipulations in signaling cues and activity in
relation to synaptic communication. Combining physiological measures of larval
CNS circuits with the anatomical profiles along with behavior assays will
undoubtedly be an area of significant interest in the next few years.
5-HT and its receptors have many physiological functions in animals including
cardiovascular physiology (Nebigil et al., 2001; Hoyer et al., 2002). 5-HT was initially
isolated from blood and found to be a vasoconstrictor (Rapport et al., 1948), making
it an attractive neurotransmitter for regulating cardiovascular functions. Loss of Tph1
gene (peripherally expressed TPH) in mice results in abnormal cardiac function
(Cote et al., 2003) and knock out of the 5-HT2B receptor in mice results in lethality
due to defects in heart development (Nebigil et al., 2000). 5-HT2A mediates arterial
vasoconstriction and 5-HT1B receptor mediates contraction of pulmonary arteries
(Hoyer et al., 1994; MacLean et al., 1996). 5-HT receptors are shown as potential
targets to treat hypertension and other peripheral vascular diseases (Frishman and
Grewall; 2000). 5-HT2B receptor is the major receptor 5-HT2 subfamily present in
heart. (Choi et al., 1997; Choi and Maroteaux, 1996; Lauder et al., 2000). In my
89
studies 5-HT2dro transgenic lines have a lower initial heart rate when compared to
that of room temperature or controls that were grown at high temperature, but
sensitivity to 5-HT is not changed. Probably in Drosophila cardiac function is not
modulated by 5-HT2dro completely. Probably other 5-HT receptors have a role in
cardiac function. Detailed pharmacological studies with agonists and antagonists of
all 4 5-HT receptors of Drosophila would give more insight to this issue.
Clinically in humans, in addition to the role of these receptors during
embryogenesis, they may be important as determinants in migraine, hypertension,
heart failure, neurodegenerative diseases and brain maturation disorders such as
schizophrenia or autistic behaviors. This study is significant since it will provide
pertinent information, which addresses the degree in synaptic performance at the
release sites over developmental stages in a model system of Drosophila when a
defined receptor subtype is knocked down in expression. The findings can be
correlated to the underlying factors of the serotoninergic system during development
which provides a frame work for future studies in knocking down the other three 5-
HT receptor subtypes or even sensitivity to other neuromodulators such as
dopamine or octopamine. Thus, there is premise that understanding the fundamental
basics of synaptic transmission in this model system will be directly relevant to all
neural systems, including humans.
90
FIGURES:
Figure 4.1: Locomotory movements at room temperature.
A) Body wall and B) mouth hook movements at room temperature. Larvae for CS,
HsZ2 and Y32 were grown at room temperature and early third instars were used for
behavior measurements. No significant difference was seen among 3 groups (N >
10, ANOVA).
91
Figure 4.2: Locomotory movements of larvae that are grown at high temperature.
A) Body wall and B) mouth hook movements at room temperature. Early 3rd instar
larvae of CS, HsZ2 and Y32 were given a heat pulse of 33oC and measurements
were carried out at room temperature. Body wall movements were significantly
reduced in transgenic lines when compared to CS (N > 25, ANOVA P< 0.05). No
significant difference was seen among 3 groups for mouth hook movements (N > 25,
ANOVA).
92
Figure 4.3: Locomotory movements at high temperature.
A) Body wall and B) mouth hook movements at 33oC. Early 3rd instar larvae for CS,
HsZ2 and Y32 were given a 4Hr heat pulse and behavior measurements were
carried out 33oC. No significant difference was seen among 3 groups (N > 10,
ANOVA) for body wall or mouth hook movements.
93
Figure 4.4: Locomotory movements at 31-32C.
A) Body wall and B) mouth hook movements were measured for larvae that were
grown 31-32oC. Early 3rd instar larvae for CS, HsZ2 and Y32 were used for behavior
measurements. No significant difference was seen among 3 groups for body wall or
mouth hook movements (N > 10, ANOVA).
94
Figure 4.5: Development curve for low temperature.
CS, HsZ2 and Y32 were grown from 1st to 3rd instar at 18oC. A) Cumulative sum for
time to pupation from eggs for CS, HsZ2 and Y32. B) Relative cumulative sum for A.
No significant difference was seen among the 3 groups (K-S test). C) Cumulative
sum for time to eclosion from pupa formation for CS, HsZ2 and Y32. D) Relative
cumulative sum for C. Time for each pupa is adjusted to ‘0’ and calculated time
taken from pupation to eclosion. No significant difference for time to eclosion was
observed for 3 groups (K-S test).
95
Figure 4.6: Development curve for room temperature.
CS, HsZ2 and Y32 were grown from 1st to 3rd instar at room temperature (21-22oC).
A) Cumulative sum for time to pupation from eggs for CS, HsZ2 and Y32. B)
Relative cumulative sum for A. No significant difference was seen among the 3
groups (K-S test). C) Cumulative sum for time to eclosion from pupa formation for
CS, HsZ2 and Y32. D) Relative cumulative sum for C. Time for each pupa is
adjusted to ‘0’ and calculated time taken from pupation to eclosion. No significant
difference for time to eclosion was observed for 3 groups (K-S test).
96
Figure 4.7: Development curve for high temperature.
CS, HsZ2 and Y32 were grown from 1st to 3rd instar 31-32oC. A) Cumulative sum for
time to pupation from eggs for CS, HsZ2 and Y32. B) Relative cumulative sum for A.
50% of CS larvae took 120 hrs for pupation whereas HsZ2 and Y32 took about 140
hrs. But very few of HsZ2 and Y32 larvae reached pupation at this temperature.
HsZ2 showed a significant difference from CS and Y32 (K-S test, P < 0.01).
97
Figure 4.8: Spontaneous activity.
The percent change in frequency per second (activity) from saline to 5-HT (100nM)
is measured in CS, HsZ2 and Y32 larvae grown at permissive temperature (A) and
non-permissive temperature (B). A) At room temperature no significant difference in
sensitivity for 5-HT 100nM was seen among the 3 groups (N=5, ANOVA). B) CS
and HsZ2 larvae that were grown at high temperature showed a decrease in activity
in presence of 5-HT (100nM) but not significant (N=5, ANOVA). Whereas Y32
showed a significant increase in activity (N = 5, ANOVA, P< 0.01)
98
Figure 4.9: Sensory-CNS-motor circuit.
A) A percent difference in the activity of motor units to muscle 6 before and after
application of 5-HT (100nM) in larvae grown at permissive temperature. All the 3
groups showed an increase in activity but no statistical difference seen (N =5,
ANOVA). B) A percent difference in activity exposed to 5-HT (100nM) for larvae that
were grown at non-permissive temperature. CS and Y32 showed an increase in
activity but Y32 showed a decrease in activity. No statistical difference seen among
the 3 groups (N=5, ANOVA).
99
Figure 4.10: Heart rate.
The percent change in heart rate upon exposure to 5-HT (100 nM). A) CS, HsZ2 and
Y32 larvae that were grown at room temperature showed an increase in heart rate
with no statistical difference (N=10, ANOVA). B) CS, HsZ2 and Y32 larvae that were
grown at high temperature showed an increase in heart rate with no statistical
difference (N=10, ANOVA). But the initial heart rate in HsZ2 and Y32 are lower
giving an abnormal % change in BPM. C) Initial heart rate for CS, HsZ2 and Y32
grown at high temperature. The initial heart rate for HsZ2 and Y32 is statistically
lower as compared to that of CS (ANOVA, P<0.0012). Also this group has a lower
initial heart rate as compared to that of room temperature raised CS, HsZ2 and Y32.
(BPM – beats per minute).
100
CHAPTER 5
DIRECT INFLUENCE OF SEROTONIN ON THE LARVAL HEART OF
DROSOPHILA MELANOGASTER
ABSTRACT
The heart rate (HR) of larval Drosophila is established to be modulated by
various neuromodulators. Serotonin (5-HT) showed dose dependent responses in
direct application within semi-intact preparations. At 1nM HR decreased by 20% and
increased at 10nM (10%) and 100nM (30%). The effects plateaued at 100nM. The
action of 5-HT on the heart were examined with an intact CNS and an ablated CNS.
The heart and aorta of dorsal vessel pulsate at different rates at rest and during
exposure to 5-HT. Splitting the heart and aorta resulted in a dramatic reduction in
pulse rate of both segments and the addition of 5-HT did not produce regional
differences. The split aorta and heart showed a high degree of sensitivity to sham
changes of saline and no significant effect to 5-HT. Larvae fed 5-HT (1mM) did not
show a significant change in HR. Since MDMA is known to act as a weak agonist on
5-HT receptors in vertebrates, I tested exogenous application however no significant
effect from 1nM to 100uM was observed in larvae with and without an intact CNS. In
summary, direct application of 5-HT to the larval heart had significant effects in a
dose-dependent manner and MDMA had no effect.
101
INTRODUCTION
The various functional aspects of heart regulation in development to normal
maintenance throughout the larval life and metamorphosis of Drosophila
melanogaster are being extensively addressed (Ashton et al., 2001; He and Adler,
2001; Molina and Cripps, 2001; Ponzielli et al., 2002; Sláma and Farkaš, 2005;
Wessells and Bodmer, 2004). In addition, regulation of the heart via hormonal and
direct neural innervation continues to be an active research area using this model
organism (Dulcis and Levine 2005; Dulcis et al. 2005; Johnson et al. 2002; Miller,
1997; Papaefthimiou and Theophilidis, 2001). Relatively recently 5-HT receptors (5-
HT1B/1CR and 5-HT2A/2BR subtypes) were shown to be present in the vertebrate aortic
and mitral valve cells of the heart (Fitzgerald et al. 2000; Roy et al. 2000) and linked
to some forms of cardiac valve disease with altered regulation (Jian et al. 2002).
Since the larvae develop quickly and the age can be precisely timed, the
development of the heart from embryo throughout growth of larval stages, as well as
through metamorphosis from larvae to adult, can be readily examined within a
matter of 1 to 2 weeks. High throughput screening is possible to asses multitudes of
pharmacological agents or mutational screens (Gu and Singh 1995).
The Drosophila heart, better known as the dorsal vessel, is a continuous tube
extending from the last abdominal segment to the dorso-anterior region of the
cerebral hemisphere. The heart is divided into anterior aorta and posterior heart
(Rizki, 1978). The heart does not pulsate constantly as noted when larvae are
preparing to crawl (Rizki 1978). When a larva is engaged in feeding the pulsation of
102
the heart and the motion of the mouth hooks are seen to be functionally related. This
is partly due to the fact that the terminal ligaments of the aorta are attached to the
pharyngeal region of the alimentary canal.
In the larvae the origin of heartbeat is known to be myogenic (Dowse et al.
1995; Johnson et al. 1997) however, the larval heart has not been shown to have
innervation to date. Where as in the adult, Dulcis and Levine (2003) have shown that
the heart is innervated. Thus, the current dogma was that the larval heart is
myogenic without innervation. Johnstone and Cooper (2006) did show that the aorta
is innervated but they did not observe the true region of heart to be innervated. (It
should be noted that this observation was noted after this chapter had been
published. Therefore some interpretations of this chapter should be considered in
regards to the published current findings, Johnstone and Cooper, 2006) The HR in
larvae can be altered by neurotransmitters and neuromodulators present in the
hemolymph (Johnson et al. 1997, 2000; Nichols et al. 1999; Zornik et al. 1999).
Injections in 3rd instars and early pupa (P1 stage, transition between larva and pupa)
of serotonin (5-HT), dopamine (DA), acetylcholine (Ach), octopamine (OA), and
norepinephrine (NE) raise the HR (Johnson et al. 1997). Effects on HR of Drosophila
peptides have also been examined (Johnson et al. 2000; Nichols et al., 1999; Zornik
et al., 1999).
In pupa, injection of 5-HT (1�M/l) caused the HR to increase by 46% from the
base line (Johnson et al. 1997). A similar study by Zornik et al. (1999) also showed,
in the wandering 3rd instar larva, that 5-HT increases HR by 111% with a
concentration of 10-5 M (10�M/l) injected into the animal. In the Zornik et al. (1999)
103
study it was determined that the adult heart is more responsive to 5-HT than the
wandering 3rd instar larvae or the pupal heart.
Injection through the larval body wall or into pupal case of biogenic amines
can cause the activation or release of many other compounds, related to stress or
injury, into the hemolymph from endogenous sources that cannot be controlled for
during the experiment. Thus, we opened the larva in a bath of physiological saline
that washes away the hemolymph to test the direct effects of compounds on the
heart within a defined saline. Since we are not aware of any attempt to clearly
determine if the larval heart is or is not innervated we assayed the effects of
compounds with and without the CNS intact. Also since exogenous application of a
neuromodulator could cause the local release of neuroendocrine compounds we
wanted to eliminate the possibility. In addition, we have shown earlier that OA, DA,
and 5-HT have direct actions on the larval CNS neural activity (Dasari and Cooper,
2004). Also in adult flies its known that the rate of stress with age is correlated with
heart failure. In addition, HR decreases with age (Wessels et al, 2004). So to avoid
the stress induced variables on HR that could occur with injections, we examined if
feeding larvae 5-HT would have an effect on heart rate in intact preparations.
Many drugs of abuse and mind altering therapeutic medicines have actions
through the serotonin-ergic system, with some altering the presynaptic reuptake
mechanisms or acting directly as an agonist/antagonist at the 5-HT receptors. One
compound of particular interest to understand is MDMA (3,4-
methylenedioxymethamphetamine, ecstasy) and its direct action on 5-HT receptors.
Invertebrates have proven to be useful on this front in studying drugs of abuse since
104
they posses physiological systems with few compounding variables and relatively
fast developmental times (Hirsh, 2001; Rothenfluh and Heberlein, 2002; Sparks et
al., 2004; Wolf and Heberlein, 2003). MDMA is a ring-substituted amphetamine and
is a widely abused drug among young people worldwide. MDMA is a potent releaser
and also reuptake inhibitor of 5-HT, DA and NE (Green et al., 2003). In humans the
acute adverse effects that are seen after MDMA ingestion are elevated blood
pressure, increased heart rate, nausea, chills, sweating tremor, bruxism probably
through actions on the autonomic nervous system (de la Torre and Farre 2004;
Green et al., 2003; McCann et al., 1996; Peroutka et al., 1988). Previously it was
demonstrated that MDMA has direct action on neuronal 5-HT receptors in an
arthropod; thus, we also examined in parallel to 5-HT's effects the action of MDMA
on the Drosophila heart with an intact and ablated CNS.
The purpose of this study is to observe the acute effects of 5-HT and MDMA
on heart rate in intact and semi-intact 3rd instar larval Drosophila preparations. In
addition, we wanted to know if an intact CNS altered the responsiveness of the heart
to these compounds possible through direct innervation of the heart or aorta.
Preliminary results of this study were presented in abstract form (Dasari et al.,
2004).
METHODS
Stock and Staging of Larvae
The common ‘wild-type’ laboratory strain of Drosophila melanogaster, Canton
S, was used in these studies. The methods used to stage fly larvae have been
described previously (Campos-Ortega and Hartenstein, 1985; Li and Cooper, 2001).
105
All animals were maintained in vials partially filled with a cornmeal-agar-dextrose-
yeast medium. Larvae of the early third instar phase were used in these
experiments. The general dissection technique and HL3 saline content has been
previously reported (Cooper and Neckameyer, 1999; Stewart et al., 1994). The HL3
saline was derived from direct measures with ion sensitive electrodes of larval
hemolymph and maintains normal function of larval neuromuscular junctions and the
CNS (Ball et al., 2003; Dasari and Cooper, 2004). In brief, the HL3 saline was
prepared in the lab from component reagents (Sigma) and contained: 1.0 mM
CaCl2.2H2O, 20 mM MgCl2, 70mM NaCl, 5mM KCl, 10mM NaHCO3, 5mM
trehalose, 115mM sucrose, and 5mM BES (N,N-bis [2-Hydoxy-ethyl] -2-
aminoethane-sulfonic acid). The HL3 was freshly controlled for pH and temperature
prior to experimentation, as the pH will drift during storage.
Heart Rate (HR) measures
With a microscopic method, the movements of the trachea or heart were used
for direct counts of HR. A microscope (adjustable zoom 0.67 to 4.5; World Precision
Instrument, FL, USA) fitted with a 10X eye objective was used for visual
observations. With visual inspection one can readily observe the heart beating or the
trachea movements as a consequence of the heart pulling on the ligament
attachments (Fig. 5.1A). The movements of the trachea are commonly used to
monitor Drosophila larval heart rate because of the clear contrast of the structures
(Dasari and Cooper, 2005; Johnson et al., 1997; Miller, 1985; White et al., 1992).
106
Semi-intact preparation
The HR was also used to examine the direct effects of MDMA and 5-HT, at
different concentrations, in exposed hearts of larvae with an intact and ablated CNS.
These semi-intact animals were of early 3rd instar that had been dissected ventrally
and pinned on four corners (Fig. 5.1B). Guts and all the visceral organs (including
the brain for deinnervation) were removed in such a way that the heart is intact and
still attached to rostral and caudal ends of the larvae. This dissection technique has
been used to directly assess pharmacological agents on the heart of Drosophila
larvae (Gu and Singh, 1995). The dissection time was 3-6 min. The preparation was
allowed to relax while bathed in HL3 saline for 3-5 min after dissection. The
heartbeats were counted for a minute at every 5 min for 20 mins, initially in saline
and then with 5-HT containing saline. Time ‘0’ is taken at the 1st min of counting in
saline. 5-HT is added to the preparation at the 7th min and a fresh dose is added
again at 12th min. The average heart beat at the 1st and 5th min for saline and at
15th and 20th min for 5-HT or MDMA was used for analysis.
The HL3 saline was carefully controlled to be at pH 7.2 since HR slows at
high pH and speeds up at lower pH (Badre et al., 2005). Gu and Singh (1995) used
pH 7.0 for the pharmacological analysis of the heart and also showed maintained
viability. During these trials the saline remained aerated by agitation of solution
through repetitively injecting saline, through a 21-gauge needle, into a beaker.
To quantify HR either direct observations were used or the images were
recorded on to VHS tapes and analyzed by a photodiode. In the cases in which the
photodiode was used, the detector (model 276-142, Radio Shack, USA) was placed
107
in the back of a black plastic 35mm film canister and the open end was held over the
region on the monitor screen in which the heart and caudal end of the larvae was
magnified. The output of the photodiode was amplified by use of an impedance
amplifier. The impedance detectors (UFI, model 2991) allowed HR to be monitored
as a measure of dynamic change in the light path across the photodiode during each
heart contraction. These signals were recorded on-line to a PowerMac 9500 via a
MacLab/4s interface (ADInstruments, Australia). Events were measured and
calibrated with the MacLab Chart software version 3.5.6 (ADInstruments) with an
acquisition rate set at 4kHz. The HR was determined by direct measures with a
window discriminator, which measured a running average of instantaneous events.
The values were then converted to beats per minute (BPM). Similar procedures in
the use of an impedance amplifier were used as described in earlier studies for
obtaining heart rates and ventilatory rates in crayfish (Dasari and Cooper 2005; Li et
al., 2000; Listerman et al., 2000; Schapker et al., 2002).
Aorta and Heart separation Wandering 3rd instars are opened as described above. HR was measured for
2 min and then heart and aorta are separated by severing them at the junction with
fine scissors. HR was measured for another 2 min before applying saline containing
5-HT.
Intact preparation
108
Early 3rd instar larvae were glued ventrally on a glass slip using super-glue in
such a way that mouth hooks are free to move. Care was taken not to glue the
spiracles so that only ventral aspect within the mid-length was adhered to the glass.
Yeast mixed in either 5-HT or water was placed over the head of the animal. The
yeast stimulated the animal to eat, thus consuming the yeast and the compounds.
The larvae were glued down and HR measured for 2 minutes with water and yeast
followed by another 15 minutes while feeding on 5-HT containing solution. Heart
beats were counted for 30 sec intervals starting initially at the time periods between
1-2 mins, 6-8 mins.
Statistics
All the groups were first tested with one-way ANOVA and followed with
Bonferroni’s posthoc analysis. Some of groups were also analyzed using Student's t-
test.
RESULTS
Semi-intact preparation
The direct exposure of the heart tube allows individual compounds to be
directly assayed on the heart without compounding variables introduced from
endogenous hormones and substance contained in the hemolymph. After pinning
the dissected larvae open, the saline bathing solution was changed to one
containing 5-HT or MDMA. Six different concentrations of 5-HT and three different
concentrations of MDMA were examined. The 1nM 5-HT exposure resulted in a
109
significant decrease in HR (Fig. 5.2A, ANOVA p<0.05, n=10) as compared to the
saline sham exposures. Saline exchange by itself results is a slight increase in HR in
some preparations but not a significant effect for all animals. However, we felt that
experimentally it was important to provide sham controls, as this is a valid issue
when examining the effects of 5-HT and MDMA in isolated heart segments (see
below). The increased concentrations of 5-HT at 10nM produced a substantially
increased HR (Fig. 5.2A, ANOVA p<0.05, n=10). However at a 100nM the increase
in HR from sham control reached a maximum effect (ANOVA p<0.05, n=10)
compared to the 1uM, 10uM, and 100uM. There was no further significant increase
at these higher concentrations compared to 100nM but all were significantly greater
in increased HR from the saline control (ANOVA p<0.05, n=10). Thus, the 5-HT
receptors were likely saturated at 100nM with only modest increases in the mean
HR for the higher concentrations. It should be noted that each preparations was
treated individually and not exposed to a series of experiments. At concentrations of
10�M 5-HT and above, the heart is in tetany and cannot relax well between beats.
This is likely the prime reason for the saturating effect in further increases of HR with
exposure to higher concentrations.
Exposure to MDMA at various concentrations did not have a significant effect
on the HR (Fig. 5.2A,B). The animals were followed for at least 20 minutes without
any notable consequences related to HR.
Since there is no known innervation from the brain to the dorsal vessel we
examined larva with the CNS left intact (Fig. 5.2A and 5.2B) and in ones where it
was carefully removed (Fig. 5.2C and 5.2D). To test whether a potentially de-
110
innervated heart showed any differences in HR compared to larvae with an intact
CNS, the dissected larvae with and without the CNS were exposed to 5-HT and the
change in HR noted. There was significant decrease in HR to 5-HT at 1nM
(Student’s t-test p<0.05, n=10) but no significant decrease with 5-HT at 10nM and
MDMA at 1uM (n=10) as compared to saline exposure. As with the intact CNS
preparations 100nM 5-HT showed a significant increase in HR (ANOVA p<0.05,
n=10, Fig. 5.2C). Comparing the differences for intact and ablated CNS the effects of
MDMA caused a decreased HR at 1uM in larvae without a CNS (Student’s t-test
p<0.05, n=10). In addition, a reversal in the effects of 10nM 5-HT in ablated CNS
preparations was observed. Without the CNS the 10nM 5-HT had a significant effect
in reducing the HR (Student’s t-test p<0.001, n=10; compare Fig. 5.2A and 5.2C for
10nM). The mean, median, 95% confidence levels and the range of the distributions
in the data sets are shown in the whisker box plots for the intact CNS (Fig. 5.2B) and
for the preparations with an ablated CNS (Fig. 5.2D).
Heart and Aorta separated
During the initial experiments when exposing the hearts with a ventral
dissection it became apparent that the dorsal vessel had a different rhythm along its
length. These regional differences in pulse rates were not observed in the intact
preparations. In addition, exposure of the entire dorsal vessel in the ventral
dissected preparations to 5-HT suggested regional differences in the actions of 5-
HT. To address this issue the heart and aorta were transected at their junction. The
rate of beating of aorta and heart was observed in saline and during exposure to 5-
111
HT (100nM). We chose 100nM concentration as this level produced a significant
increase in HR from saline without inducing a tetanic state of the heart in whole
dorsal vessel. MDMA was not used in these preparations, as it didn’t show any
effect in direct exposure to the heart.
The number of beats within the aorta and heart were measured prior to
transection and then again after a saline wash or a saline wash containing 5-HT.
Comparisons were made by determining a percentage difference for the separated
aorta and heart to the intact state of the aorta and heart. The effect of direct
exposure of 5-HT (100nM) produced a substantial increase in HR (Fig. 5.3A,
ANOVA p<0.05, n=10) in the heart when the dorsal vessel is left intact. However
after transection the preparation became very sensitive to changes in the bathing
media. When sham controls for mechanical disturbances on the heart rate were
compared to saline changes containing 5-HT, there is no significant effect of 5-HT.
Saline exchange alone increased the rate of the aorta significantly (Fig. 5.3B,
ANOVA p<0.05, n=14). A small effect was also produced on the true heart with
saline exchange (Fig. 5.3B, ANOVA p<0.05, n=14). The exposure to 5-HT also had
a substantial effect on the rate of both the heart and aorta but not significantly over
the sham control effects (n=7). Therefore, we concluded that 5-HT did not show any
differential effects on the rate of the two segments when the dorsal vessel segments
were isolated from each other.
Intact Preparation
112
In examining the possibility of 5-HT gaining access to the hemolymph and
altering HR, we fed early 3rd instars 5-HT (1mM) mixed with yeast. Control
experiments of water and yeast were also conducted for the same period of time.
The controls maintained a steady HR for 8-10 minutes, however the larvae eating a
5-HT tainted diet after 6-8 minutes dropped in HR for some larvae but not others.
The change was not significant (Student's t-test) for 5-HT fed larvae compared to
controls. The same larvae feeding on 5-HT were followed further for 12-15 minutes,
and in some preparations HR was not noticeable even though the larvae were alive
as indicated by mouth hook movements. The cardiac arrest was not due to tetany of
the heart. In other larvae no change in HR occurred before or after 15 minutes of
eating the 5-HT containing food. Thus, there was a large variation observed in the 5-
HT fed larvae as compared to controls.
DISCUSSION
In this study we showed that the larval Drosophila heart can be examined in a
semi-intact state in which the hemolymph can be exchanged with saline in order to
reduced compounding variables of endogenous neuromodulators or induced release
due to systemic injections of pharmacologic compounds. The minimal HL3 saline
maintains the HR, thus pharmacological assays can be run with an intact or ablated
CNS for high output screening as to direct actions on cardiac function. The larval
heart in a semi-intact state (heart and aorta connected) decreased its rate at 1 nM
5-HT but increased in rate with exposure higher than 10nM with a plateau in the
enhancement at 100nM. Ablating the CNS resulted in a decline in HR for 5-HT
113
exposure at 10nM however increased for higher concentrations. Separating the
dorsal heart tube into the true heart and aorta while exposure the segments to 5-HT
or saline both resulted in an increase HR with no discernable effects to 5-HT alone
over the sham effects of changing the saline bath. It is of interest to note for future
pharmacological investigations that the heart is very sensitive to exchange of the
bathing media. This was also noted in earlier studies in examining the effects of pH
on HR (Badre et al., 2005). Lastly, 5-HT introduced in the diet at a high
concentration of 1mM did not alter HR significantly but did result in an increased
variability in HR, thus suggesting feeding larvae this particular biogenic amine to
address effects on HR might not be feasible.
Since direct exposure of the heart tube to 5-HT resulted in biphasic changes
in HR with low concentration decreasing HR while higher concentrations increased
HR beacons the possibility of different 5-HT receptor subtypes with varying degrees
of binding affinity and/or activated second messenger cascades on the heart tissue
(Johnson et al., 2002). It is known that the Drosophila genome contains 4 different 5-
HT receptor-coding sequences (Colas et al., 1995; Monastirioti, 1999; Saudou et al.,
1992; Witz et al., 1990). However, posttranslational modifications or regulated RNA
editing of the 5-HT receptors in Drosophila might occur as known in vertebrates for
some 5-HT receptors subtypes (Paupard et al., 2000; Slominski et al., 2003; Visiers
et al., 2001). Differences in RNA editing is also a possibility over the development of
the larvae as shown for different types of receptors in mammals (Lee et al., 2001),
thus one should consider pharmacological and molecular analysis at various life
stages for a complete assessment in subtypes of 5-HT receptors. In this study we
114
only examined early 3rd instars, which predominantly express the 5-HT2dro receptor
subtype (Colas et al., 1995), however previous studies did not specifically address
expression in heart tissue developmentally. A complete pharmacological profile of
agonists and antagonists to 5-HT receptors would be advantageous in helping to
address the receptor subtypes in cardiac modulation within Drosophila. Although
one should also be careful in assuming the vertebrate pharmacological profiles
correctly characterizes the invertebrate systems (Sparks et al., 2003). Such
differences might explain why the heart is not sensitive to MDMA but that the larval
CNS is very sensitive to exogenous application (Dasari et al., 2004).
A number of studies have addressed the effects of biogenic amines and
peptides on cardiac function in larval and pupal stages (Johnson et al. 1997, Zornik
et al., 1999). The pupal stage offers the advantage in that it does not require to be
restrained. Even the larval heart can be viewed in freely wandering larvae but it is
considerable harder to maintain a good assessment of heart function continuously
(Dasari and Cooper, 2005). Thus, gluing or taping larvae have been preferred
methods to restrain the animal for viewing of the tracheal movements or the heart
directly. The introduction of pharmacological agents has also been preferred by
injection through the cuticle into the hemolymph. Although such methods have
certain advantages one is not confident to the effects of the stress in relation to the
animal dumping various endogenous biogenic amines and peptides into the
hemolymph. One also needs to consider heat stress is known to alter biogenic
amine levels in Drosophila (Hirashima et al., 2000) In addition circadian patterns of
5-HT are known to occur and could alter the responsiveness to exogenous
115
applications (Fowler et al., 1972). This was one reason that we chose to dissect the
larvae and expose the heart directly to 5-HT in a defined physiological saline at a
given temperature and period of day (12:00-18:00). This semi-intact preparation also
offers the advantage to introduce various compounds over time and wash out
previously introduced substances (Gu and Singh, 1995). An additional advantage of
the dissected preparation that has not previously been addressed is the fact that the
CNS can be left intact or removed. Leaving it intact and using a small volume of
bathing media one can address if compounds from the CNS can be induced for
release for examining an effect on cardiac performance. In addition if any neuronal
connections do exists on the larval heart they can be removed or left intact for
assaying various exogenous agents. Earlier studies in which TTX was bathed on the
exposed heart did not produce any significant alterations in HR (Gu and Singh,
1995) and thus it was assumed that the larval hearts are myogenic but this does not
prove that the hearts are not modulated by neuronal innervation as for mammals.
Since we did note that difference do occur in sensitivity to 5-HT in preparations with
an ablated CNS, this suggests that there may indeed be a neural input that is
modulated by 5-HT possibly at presynaptic nerve terminals on the heart as for
skeletal muscles in crustaceans (Southard et al., 2000). The activity of the CNS
circuitry in larvae is known to be influenced substantially by 5-HT as well as by
octopamine and dopamine (Dasari and Cooper, 2004). This issue of innervation of
the heart tube remains to be addressed anatomically in larval Drosophila.
Earlier reports have shown that injection of 5-HT into 3rd instars and P1 pupal
stage of Drosophila melanogaster Oregon R and Canton S strains respectively,
116
causes an increase in HR (Table 5.1). In this study, we have shown that direct
exposure of heart to 5-HT in 3rd instars of Canton S strain also causes it to increase
at high concentrations but at low concentrations of 1nM it decreases. In the previous
concentration ranges examined 5-HT produced a larger increase in HR than what
we report for semi-intact preparations. The possibility of synergistic actions of
endogenous compounds with the injected 5-HT is one possibility for enhanced
effects of 5-HT. As shown in crustaceans and leech, 5-HT has different effects in the
presence of octopamine as compared to 5-HT exposure alone (Djokaj et al., 2001;
Mesce, 2002). Given that there are many active substances contained in the larval
hemolymph, the biogenic/peptide cocktails remains an interesting avenue for further
research in isolated preparations. On the other hand the dissection of the larvae and
exposure to HL3 physiological saline might not provide the correct ionic environment
for cardiac function. The HL3 saline was originally developed and assayed for
skeletal neuromuscular transmission, however the ions were determined from larval
hemolymph samples (Stewart et al., 1994).
The ability to feed larval and adult Drosophila compounds to alter
endogenous neuromodulators has been used with success (Neckameyer et al.,
2001) but this approach is likely not feasible for readily degraded compounds by an
acidic environment or that are photo liable such as 5-HT (Lisi et al., 2003; Strawn et
al., 2001). Saliva of adult Drosophila contains enzymes known to digest chitin as
well as amylose and cellulose (Gregg et al., 1990), thus possibly larvae also contain
such abilities. Thus, we are not convinced that feeding the larvae food tainted with 5-
HT (1mM) resulted in any significant change in hemolymph levels since injection into
117
larvae or direct application on to the heart at lower concentrations resulted in a
change of HR.
Much to our dismay the transected heart tube into the true heart and aorta
became very sensitive to any alterations in changing of the bathing media which
made it difficult to assay differential effects of the aorta and the true heart to
exposure of 5-HT. It is established that the heart tube contains pacemaker activity
but the extent and control of a major pacemaker in the heart and aorta segments
has not been addressed in relation to biogenic modulation. Transecting the heart
tube into the true heart and the aorta resulted in the two segments beating at their
own intrinsic rates. The aorta significantly slowed down and the true heart increased
in rate. Thus, uncoupling the caudal-anterior communication within the heart tube
had some effect on possible unmasking some feedback or inhibition of rate in the
intact true heart where the lack of the master pattern activity of the heart on the aorta
resulted in the slower aorta pacemaker activity, which is analogous to mammalian
hearts among the pacemaker regions. Possibly after the heart tube is separated
there is a reduction in filling the aorta or less peripheral resistance for the heart
between pulses which could then effect myogenic stretch related activity. It is also
conceivable that the micro-environments in the cut vesicles related to perfusion of
the saline to luminal surface of the vessel is altered which might explain the
heightened sensitivity to saline exchange and exposures to 5-HT.
Developing a means to introduce agents without causing mechanical
disturbance of the bathing media will help in examining localized effects of
pharmacological agents on the true heart and the aorta separately. We are currently
118
examining explant cell culture of Drosophila heart tissue to determine its feasibility.
Electrophysiological recordings would give an insight into the ionic properties of the
pacemakers and the mechanistic effects of the various known modulators on the
Drosophila heart.
119
Table 5.1 Effects of 5-HT on HR as percent difference from base line as reported in various
studies for comparison.
1mM 100�M 10�M 1�M 0.1 �M 0.001 �M Referenc
e
Injection
3rd instar
� 120
± 6
� 123
± 5
� 111
± 4
NS - - Zornik et
al., 1999
Injection
P1 stage
� 75 � 55 � 28 � 46 � 15 - Johnson
et al.,
1997
Direct
application
3rd instar
- � 38
± 5
� 29
± 4
� 27
± 3
� 31
± 5
�17
± 3
This
study
The effect of 5-HT on heart rate. The percent difference in heart rate from the base
line after injection of 5-HT as reported in earlier work is shown. Zornik et al. (1999)
injected different concentrations of 5-HT in 3rd instar larva. Johnson et al. (1997)
injected 5-HT into P1 stage, the transition stage between 3rd instar and pupa.
120
FIGURES:
Figure 5.1: Dorsal Vessel.
A) Dorsal view of an intact 3rd instar larva. The movement of trachea due to pulling of
the attachments from heart is used to observe the heart rate. B) Ventral dissection of
a 3rd instar to view the heart directly. Pinning of the animal on its back after
dissection is used to directly apply the compounds on the heart with or without the
CNS intact. Small arrow indicates where the heart and aorta are separated for
transected dorsal vessel studies (see below). Tr, Trachea; Sp, Spiracles; H, Heart;
AO, Aorta.
121
Figure 5.2: Heart rate of 3rd instar semi-intact preparations.
A) % change in heart rate from saline per minute in innervated preparations (brains
attached). B) 95% confidence intervals for the same data set as in A. C) % change
in the heart rate from saline in deinnervated preparations (CNS ablated). D) 95%
confidence intervals for the same data set as in C. BPM, beats per minute. A star
denotes ANOVA, P<0.0001, followed by Bonferroni test, P< 0.05.
122
Figure 5.3: The effects of 5-HT on intact and on the isolated heart and aorta
segments.
The number of beats were measured before and after severing the dorsal vessel at
the aorta and heart junction. A) Percent change in the heart rate from saline to
exposure to exogenous 5-HT (100nM) in an intact dorsal vessel. B) The beat rate
was measured in aorta and heart separately before and after dividing the vessel. A
percent change is calculated for intact to split aorta and heart in saline (n =14). The
percent change in HR before and after saline wash in the split aorta and heart
preparations revealed a significant increase in rate (n=14; ANOVA, P< 0.0002,
Bonferroni test, P< 0.05). Exposure to 5-HT (100nM) produced a larger mean effect
but there was also considerable variation among the preparations, thus no trend to
5-HT exposure was observed as compared to the sham saline effects (n=7).
123
CHAPTER 6
DISCUSSION
In these studies I have shown that the neuromodulators 5-HT, OA and DA
have a role in modulating the CNS in larval Drosophila melanogaster. In order to
examine the effect of these neuromodulators on the larval CNS I developed an in
situ preparation consisting of an intact sensory-CNS-motor central circuit in which
exogenous applications could be applied. All three neuromodulators resulted in an
increase in the firing frequency in the circuit; however, exogenous application of OA
and DA at NMJ caused a decrease in EPSP amplitudes where as 5-HT showed no
effect. Also, I have shown that 5-HT modulates heart rate (HR) in Drosophila larva.
Cardiac activity is an important physiological function that I used in parallel in
addressing issues related to manipulating the levels of 5-HT in the whole animal as
well as 5-HT receptor subtypes. The heart 5-HT assay serves as an additional tissue
to examine along with CNS function. The modulatory effect on the sensory-CNS-
motor circuit and the potential relationship on behavior, synaptic growth and
plasticity interested me in pursuing their role on developmental processes in larval
Drosophila. Many drugs of abuse such as cocaine, amphetamine and MDMA
(ecstasy) are known to alter the serotonergic system in mammals. These drugs also
have the potential of effecting synaptic transmission, behavior and development.
In vertebrates, 5-HT is known to play a role in development, physiological
process, behaviors, and to be associated to many pathological diseases. In this
study I have shown, when 5-HT is depleted in Drosophila larvae by feeding p-CPA,
the developmental rate is retarded. Also, the percent of death from 1st to pupal stage
124
is (p-CPA) dependent of 5-HT. Hence, 5-HT plays a very important role in larval
Drosophila development. Locomotor behavior (body wall movements) and mouth
hook movements are reduced in larvae fed p-CPA. These behavioral responses may
in part be accounted for by the decreased responsiveness of the sensory-CNS-
motor circuit to exogenous application of 5-HT in p-CPA fed animals. Since there is
a reduced responsiveness to exogenous applications it is likely that synaptically
released 5-HT would also produce a reduced response. However the detailed action
of 5-HT on the neural circuits those at are responsible for eating and crawling
behaviors are not known.
Unlike the p-CPA fed larvae, exogenous application of 5-HT on sensory-CNS-
motor circuit in larvae fed MDMA showed a significant increase in sensitivity to 5-HT.
Also MDMA fed larvae showed a significant decrease in locomotor movements but
no alteration in feeding behavior. Such a differential effect helps one in dissecting
the pharmacological differences among these behavioral circuits. If one knew the
specific action of MDMA such as binding affinity for the particular 5-HT receptors
and transporters that MDMA targets then one could propose neuronal wiring
diagrams for these differences in behaviors. In humans MDMA causes a euphoric
sensation, elation and a state of stimulation. MDMA causes reversal of 5-HT
transporter and release of 5-HT. Another drug available in market, fluoxetine
(Prozac), a selective 5-HT uptake inhibitor, is used as an antidepressant. Fluoxetine
blocks the 5-HT transporter from re-uptaking 5-HT from synapses. Though both
these drugs increase concentration of 5-HT at synapses they show opposite effects.
This could be because of various effects of MDMA just not only on 5-HT system but
125
also other neurotransmitter like dopamine, norepinephrine, acetylcholine. Also
fluoxetine inhibits MDMA or its metabolites entry into presynaptic terminal, thus
giving its neuroprotective effects against MDMA (Sanchez et al., 2001). Using
behavior studies along with physiological studies such distinctions can be made. I
had assumed that the Drosophila model could offer some insight into these matters,
but it appears that MDMA does not deplete 5-HT from nerve terminals.
Altering 5-HT function by changing the target instead the production end was
another approach that I undertook in these studies. I was fortunate that fly strains
were made in which the 5-HT receptor (5-HT2dro) can be reduced in expression by
an antisense approach (Chapter 3). In these strains 5-HT receptor knock downs
affected both growth and locomotor movements. I also discovered that even at room
temperature the heat shock induced antisense production might be activated since
development was slowed as compared to when the strains were raised at 18oC.
With a significant knock down in the receptor expression from 1st to 3rd instar stage,
growth and the sensitivity to 5-HT in the central circuits were also affected. Hence I
showed that 5-HT as well as 5-HT2dro receptor plays an important role in the
development of Drosophila melanogaster.
The results I obtained, in regards to p-CPA treatments, are similar to those for
other animals. Koe and Wiessman (1966) showed that administration of p-CPA to
rats decrease their brain 5-HT levels and when administered to pregnant rats,
slowed down the neuronal differentiation in brain regions of pups containing 5-HT
terminals or known to have high 5-HT content in adults (Lauder and Kerbs, 1978).
Even pregnant rats exposed to p-CPA on a critical period for CNS development
126
resulted in a decrease in 5-HT in embryonic neurons, showing that the maternal p-
CPA treatment effects the development of the embryo (Butkevich et al., 2003). The
mRNA for mMany of the 5-HT receptors mRNA are present throughout the
development process of the brain in many animals (Whitaker-Azmitia et al., 1996;
Hellendall et al., 1993). Thusis is probably why a, deficiency in 5-HT effects brain
development in animals and in some cases the consequences are seen in the
postnatal life of the organism. This is also very important with antipsychotics related
to the 5-HT axis since no standards are being followed for breast-feeding mothers
and potential effects on neonatal development.
It is possible that a reduction in neural activity either by alterations in
dopamine or 5-HT could reduce the growth related hormone producton and/or
release. A reduction of ecdysone production can prolong larval stages for a week (Li
and Cooper, 2001). The neural regulation for feedback/release of ecdysone and
juvenile hormone is well established (Gu et al., 1996; Song and Gilbert 1996).
Neural factors like PTTH stimulate prothoracic gland to stimulate and release
ecdysone which is later converted into a usable form, 20-hydroxyecdysone, in other
tissues. The conversion to 20-hydroxyecdysone is regulated by inositol 1,4,5-
triphosphate (InsP3) and cAMP signaling pathway (Venkatesh et al., 2001). In one of
the 5-HT signaling pathways both InsP3 and cAMP act as 2nd messengers. Hence
altered levels of 5-HT might effect the release/ secretion of ecdysone.
My study is the first to quantitatively show, by HPLC, how much 5-HT is
reduced by feeding p-CPA to larval Drosophila. It is interesting to note that the
reduction was observed for the CNS but not for whole larva. It is not surprising since
127
the CNS may only contain a small amount in respect to the whole body. Studies in
mice have shown 2 distinct isoforms of TPH (Walther et al., 2003), TPH 1 and TPH
2. TPH 1 is expressed in the peripheral regions (non-neuronal) and is scarce in the
CNS. On the other hand, TPH 2 is expressed only in brain. Zhang et al., (2004)
showed that TPH 2 is the main source for 5-HT synthesis is brain. Recently in
Drosophila, it is shown that there are two different forms of TRH enzyme, one that is
specific to the brain, called as DTRH, and another (DTPH, Drosophila tryptophan-
phenylalanine hydroxylase) is in the periphery (Coleman and Neckameyer, 2005).
So perhaps p-CPA selectively targets a DTRH in Drosophila. Also another
significant finding from the studies utilizing HPLC was that 5,7-DHT did not result in
a drop of 5-HT production in the CNS and therefore the 5-HT producing neurons
were probably not killed out. Such a finding stresses the importance of not extracting
pharmacological phenomena that is well established in vertebrates and assumes
that the same approach will work in an invertebrate species.
5-HT is involved in motor unit co-ordination in other animals, including
invertebrates, so it is not so surprising that some effects in bodily movements are
observed in Drosophila related to 5-HT (Sewell et al., 1975; Kamyshev et al., 1983).
For example, leech swimming behavior is modulated by 5-HT (Kristan and
Nusbaum, 1982). Central pattern generators involved in chewing and respiration are
also affected (Jacobs and Fornal, 1993). Likewise, P-CPA administered
intraperitonally in rat pups showed a marked decrease in their growth, posture and
locomotor activity (Myoga et al., 1995). Of course to compare studies one has to be
cognizant of dosage and scaling between animals for body size but also for
128
metabolic rates (White and Seymour, 2005; West et al., 2002). p-CPA (10mg/ml) fed
to adult Drosophila flies showed a decrease in their ability to fly (Banerjee et al.,
2004) but their locomotor activity increases when treated with 150 ug/ml (Kamyshev
et al., 1983). This difference observed in adults and larvae in the action of p-CPA
might be because of the developmental changes. The circuits in the larval fly system
are also different from adult flies. Given that the larval CNS is reorganized as
compared to the adult CNS it would be expected that locomotion between the
different stages would differ in response to 5-HT as well as eating behaviors. Colas
et al., (1999) also showed that the 5-HT receptor subtypes even vary over the larval
stages, so I would expect that locomotion as well as eating responses might vary
differentially at the different stages to such pharmacological manipulations as
feeding p-CPA. Studies of this nature have not been undertaken yet, but are worthy
of investigation.
Also p-CPA is not a specific inhibitor of TPH but likely also inhibits
phenylalanine hydroxylase (Koe and Weissman, 1966; Miller et al., 1975, 1976), the
rate-limiting enzyme in tyrosine synthesis (Neckamayer and White, 1993). In my
HPLC study, I found a small decrease in the levels of DA in brains of p-CPA fed
larvae. Hence the behavioral responses we measured may not be completely due to
5-HT depletion, but depletion of DA or even a combinational effect of the two being
reduced. Even in mammals, effects of p-CPA on locomotor activity are shown to be
divergent in reducing activity (Fibiger et al., 1971; Pirch, 1969) to enhancing
movements (Borbely et al., 1973; Marsden et al., 1976). These differences seem to
be based on the dosage of p-CPA and the environment in which the experiments are
129
carried out (Dringenberg et al., 1995). Also p-CPA can cause a reduction in food
intake (Borbely et al., 1973; Marsden et al., 1976). Besides 5-HT is present in
intestinal tissue and helps in gastrointestinal function so a reduction could alter
dietary/digestion factors. The alteration in 5-HT level affects the mobility of the gut
and is associated with irritable bowel syndrome (Crowell, 2001). The larvae treated
with p-CPA were smaller in size as compared to controls and this might be a part of
the developmental retardation seen because reduced food intake and absorption.
MDMA, in mammals is known to work through the serotonergic system
(Green et al., 2003). Also it affects dopamine (DA) and acetylcholine (Ach) function,
which can then result in very broad actions in the CNS. Feeding MDMA to
Drosophila larvae caused a delay in development, in a dose dependent manner. The
higher the dose the greater the delay in development. However in rats that were
administered MDMA (Day 11-20, thisat is similar to thehuman late third trimester
stagein humans; Broening et al., 2001) no effect on survival was observed but
spatial and sequential learning and memory was reduced (Broening et al., 2001).
Since MDMA did not deplete 5-HT in the larval CNS, as noted byin the HPLC
studies, one would assume that MDMA is not functioning in the same manner in
Drosophila as in mammals. Drosophila 5-HT transporter (dSERT) is homologous to
human and rat SERT by only 51%. Demchyshyn et al. (1994) reported that dSERT
is not similar to the hSERT pharmacologically. The affinity of dSERT is different for
antidepressants like imaprimine as compared to hSERT. Thus, MDMA may not be
acting on dSERT the same way as on hSERT. It would be interesting to compare,
possibly with radiolabel binding assays, if indeed the 5-HT transporter in fly neurons
130
have a different binding affinity for MDMA than that of vertebrates. Possibly the
molecular sequence would reveal substantial differences in the binding domains to
account for the differential effect among species.
The acute effects of administration of MDMA in rats immediately results in an
increased locomotion (Callaway et al., 1990). This hyperactivity is based on the
release of 5-HT by the transporter working in reverse (Callaway et al., 1990). The
dumping of 5-HT in neurons is reduced when rats are treated with p-CPA (Callaway
et al., 1990). 5-HT1B (Callaway et al., 1990; Rempel et al., 1993) and the 5-HT2A
receptors (Kehne et al., 1996) were shown to be activated by MDMA and
hyperactivity in rats is dependent on the activation of these receptors. But in
Drosophila larvae fed MDMA from 1st to 3rd instar stage no increase was observed
but instead a decrease in locomotor movements occurred. An aspect that I did not
investigate was the potential variation due to gender. Recently Palenicek et al.,
(2005) showed that female rats are more susceptible to acute locomotor stimulating
effects of MDMA than male rats. All the other effects of MDMA were seen to be
similar in male and female rats (Walker et al., 2006). Such sexual differences in the
effects of MDMA in Drosophila larvae as well as in adults could be followed up in
future studies.
Just as 5-HT modulates heart rate in humans by either increasing (Harris et
al., 1960) or decreasing (Hollander et al., 1957) the rate depending on the dosage,
5-HT has a pronounced effect on the heart rate in larval Drosophila,; however, the
effect is directly on the heart tissue. MDMA also causes an increase in the heart
rate and blood pressure in humans (Freedman et al., 2005; Mas et al., 1999);
131
however, in rats a decrease in heart rate was observed at a dose of 1 mg/Kg
(O’Cain et al., 2000) and at a dose of 20mg/kg., Aa dose considered to be
neurotoxic produced intense bradycardia (O’Cain et al., 2000). 5-HT is involved in
cardiovascular function in humans as well as insects (Chapter 3) and their close
cousins the crustaceans (Listerman et al., 2000). 5-HT2B receptors are present on
valves, and defects in these receptors result in valvular cardiopathies in mammals
(Setola et al., 2005; Kaumann and Levy, 2006). It is known that a reduction in 5-HT
synthesis by a loss of TPH1 in mice can produce abnormal heart rate and lead to
heart failure (Cote et al., 2003). In my studies, exogenous application of 5-HT
caused an increase in heart rate in a dose dependent manner with the slightest
effect being noted at 10nM. Interestingly MDMA did not have any significant effect
on the Drosophila larval heart rate at doses ranging from 1µM to 100µM. P-CPA fed
Drosophila larvae also did not show any difference in the heart rate as compared to
that of controls. This might be because 5-HT levels were not reduced in the whole
larvae feed p-CPA, so the heart might have been exposed to normal levels of 5-HT
throughout the feeding regimen of p-CPA. In addition, it is not known if MDMA binds
to Drosophila 5-HT receptors. Recently it was shown by my colleague, Dr. Andrew
Johnstone, that the aorta of larval heart is innervated (Johnstone and Cooper, 2006);
however, the effects of 5-HT on this innervation has not been investigated. Dulcis
and Levine (2003) showed, in adult Drosophila, that the heart is innervated and that
the nerve terminals are glutamatergic, but they did not investigate neuromodulation
of this innervation. The mechanism of 5-HT's action on the Drosophila larval heart is
not known since the receptor subtype has not been investigated yet.
132
Pharmacological studies are underway in the Cooper laboratory. Once the receptor
subtypes are known then logical predictions of why MDMA does not produce a direct
effect on the larval heart as compared to the CNS can be made.
Neural circuits are conserved across phylogenetic orders. For example,
central pattern generators of locomotion in spinal cords are conserved from lamprey
to rats (Katz and Harris-Warrick, 1999). Formations of various neural circuits are
known to be dependent on signaling cues from neurotransmitters and
neuromodulators. Neurotransmitters and neuromodulators are expressed in high
amounts during certain stages of developmental periods as compared to others,
which is key to their role. Neuromodulators can regulate critical periods of a
development with altered stimulation when required for correct development within
given developmental windows.
Hubel and Wiesel (1970) showed that there is a critical period for effects of
visual deprivation. Critical period of monocular deprivation in kittens is at 3 and 4
week of age in layer IV and slowly shifts to other extragranular layers at 6 weeks and
end at 1 year of age (Hubel and Wiesel, 1970; Daw et al., 1992). The sensitivity of
the visual system in binocular competition is regulated by extracellular inputs like
serotonergic and cholinergic system (Kojic et al., 2001). 5-HT has been proposed to
be a factor that determines critical period for synaptic plasticity in the rat visual cortex
of rats (Edagawa et al., 2001). 5-HT is shown to increase with development and
suppress induction of long-term potentiation (LTP) in rat visual cortex (Edagawa et
al., 2001). Similarly 5-HT has been shown to be involved in critical periods in the
development of other neural circuits. Treatment of 9-day pregnant rats with p-CPA,
133
critical day for normal serotonergic system development in fetus, caused alteration
5-HT levels and also decreased the sensitivity of pain in offspring (Butkevich et al.,
2003). In insects like the bee (Bombus terrestris), the 2nd larval stage is the critical
period for caste determination (worker and queen bee). During this critical period,
juvenile hormone (JH) biosynthesis is eight to ten times higher in queen larvae than
worker larvae (Cnaani et al., 2000). In Drosophila, abolishing activity of acetylcholine
esterase enzyme (AChE) during embryogenesis is lethal showing that there is a
critical period in CNS during embryogenesis where AChE function is required
(Greenspan et al., 1980). I have also shown that 5-HT is important during the larval
development of Drosophila. In addition, Colas et al (1999) did show that 5-HT2dro
receptor function is important during embryonic development. As a future step, using
pharmacological manipulations and genetic tools during critical periods, where 5-HT
signaling is important during development, will help to advance the understanding of
neural development.
The sensory input controls CNS and motor unit development and function.
Invertebrate models like Tritonia (mollusk), somatogastric ganglion (STG) of
crustaceans, have proven to be very useful in learning about rhythmic activity. 5-HT
modulates CPGs in both vertebrates and invertebrates. STG of crustaceans is
shown to be modulated by biogenic amines (Marder and Thirumalai, 2002). In
Tritonia dorsal swim interneurons (DSIs) are a set of serotonergic neurons that are
intrinsic to swim CPG (Katz et al., 1994). Fickbohm et al. (2000) showed that
increasing 5-HT in Tritonia enhances the ability of DSIs to elicit rhythmic activity in
swim interneurons but also DSIs were silent or less active in between bouts of
134
activity. In isolated lamprey spinal cord, a vertebrate model, an increase in 5-HT
reduces fictive swim burst frequency (Harris-Warrick and Cohen, 1985). In
Drosophila the pharyngeal muscles are also involved in feeding and it is known that
exogenous application of cholinergic agonists in a 3rd instar semi-intact preparation
induced bursting activity in muscles (Gorczyca et al., 1991). 5-HT is also present in
pharyngeal muscle fibers (Valles and White, 1986) and exogenous application of 5-
HT also initiates activity in these muscles (Budnik and White, 1987b). Suster and
Bate (2002) showed that though the circuit for peristalsis and crawling is formed in
embryos and larvae of Drosophila in complete absence of sensory input, the actual
patterns of locomotion and its integration into the circuit is lost. In 3rd instar semi-
intact larval preparation I have shown that 5-HT (10µM) increases motoneuron firing
frequency in a biphasic manner. Decreasing 5-HT levels during development
decreased this firing frequency. Octopamine (OA) at 10µM also caused a rhythmic
movements in semi-intact preparation and caused an increase in firing frequency so
it is not only the presence of the compounds but at what concentration. The varied
concentrations at particular periods in develop could be crucial but as far as I am
aware no one has investigated this topic. Recently, OA was shown to modulate
CPGs for larval locomotion (Fox et al., 2006) so it would be of interest to see the
long term consequences of excess or depletion of OA on the CPGs.
There are many continuation projects that have stemmed from these studies,
many of which I have already highlighted above. Some of the tangible projects that
would be immediately beneficial to the fileld in modulation of neural development are
outlined next. The effects of the 5-HT deficiency during larval development can be
135
extended to examine the consequences on neural development in the adult CNS. 5-
HT is known to be abundant in adult eyes (Rodriguez Moncalvo and Campos, 2005)
and involved in learning/memory (Tempel et al., 1984; Alshuaib et al., 2003).
Deficiency of 5-HT during larval and pupal development might cause defect in the
formation of 5-HT nerve terminals within the adult CNS. Testing vision, learning and
memory in adult flies, as well as in larvae, after various bouts of p-CPA feeding
would help us in further understanding the role 5-HT in development and modulation
of these behaviors. The role of the 5-HT receptors in these same behaviors of
course could parallel such studies in the production of 5-HT. Many studies have
shown Drosophila as model organism for discovering the mechanisms of action of
drugs of abuse like, cocaine. Such additions to the reasons why MDMA resulted in
behavioral differences while not depleting the 5-HT in the CNS is of interest.
136
REFERENCES
CHAPTER 1 Aghajanian, G.K., Marek, G.J., 1999. Serotonin and hallucinogens.
Neuropsychopharmacol. 21, 16S – 23S.
Andresen, V., Camilleri, M., 2006. Irritable bowel syndrome: recent and novel
therapeutic approaches. Drugs. 66, 1073 – 1088.
Azaryan, A.V., Clock, B.J., Rosenberger, J.G., Cox, B.M., 1998. Transient
upregulation of mu opioid receptor mRNA levels in nucleus accumbens during
chronic cocaine administration. Can. J. Physiol. Pharmacol. 76, 278 – 283.
Bae, S., Zang, L., 2005. Prenatal cocaine exposure increases apoptosis of neonatal
rat heart and heart susceptibility to ischemia-reperfusion injury in 1-month-old rat. Br.
J. Pharmacol. 144, 900 – 907.
Baker, M.W., Croll, R.P., 1996. Modulation of in vivo neuronal sprouting by serotonin
in the adult CNS of the snail. Cell Mol. Neurobiol. 16, 561 – 576.
Baker, M.W., Vohra, M.M., Croll, R.P., 1993. Serotonin depletors, 5,7-
dihydroxytryptamine and p-chlorophenylalanine, cause sprouting in the CNS of the
adult snail. Brain Res. 623, 311 – 315.
137
Banerjee, S., Lee, J., Venkatesh, K., Wu, C.F., Hasan, G., 2004. Loss of flight and
associated neuronal rhythmicity in inositol 1,4,5-trisphosphate receptor mutants of
Drosophila. J. Neurosci. 24, 7869 – 7878.
Barnes, N.M., Sharp, T. (1999) A review of central 5-HT receptors and their function.
Neuropharmacol. 38, 1083 –1152.
Bar-Peled, O., Gross-Isseroff, R., Ben-Hur, H., Hoskins, I., Groner, Y., Biegon A.,
1991. Fetal human brain exhibits a prenatal peak in the density of serotonin 5-HT1A
receptors. Neurosci. Lett. 127, 173 – 176.
Barron, A.B., Schulz, D.J., Robinson, G.E., 2002. Octopamine modulates
responsiveness to foraging-related stimuli in honey bees (Apis mellifera). J. Comp.
Physiol. A Neuroethol. Sens. Neural. Behav. Physiol. 188, 603 – 610.
Bolonna, A.A., Arranz, M.J., Mancama, D., Kerwin, R.W., 2004. Pharmacogenomics-
-can genetics help in the care of psychiatric patients? Int. Rev. Psychiatry. 16, 311 –
319.
Broening, H.W., Morford, L.R., Inman-Wood, S.L., Fukumura, M., Vorhees, C. 2001.
3,4-Methylenedioxymethamphetamine (ecstasy)-induced learning and memory
impairments depend on the age of exposure during early development, J. Neurosci.
21, 3228 – 3235.
138
Burns, C.M., Chu, H., Rueter, S.M., Hutchinson, L.K., Canton, H., Sanders-Bush, E.,
Emeson, R.B., 1997. Regulation of serotonin-2C receptor G-protein coupling by
RNA editing. Nature. 387, 303 – 308.
Casanueva, F.F., Villanueva, L., Peiialva, A., Cabezas-Cerrato, J., 1984. Depending
on the stimulus, central serotoninergic activation by fenfluramine blocks or does not
alter GH secretion in man. Neuroendocrinol. 38, 302 – 308.
Chang, D.J., Lim, C.S., Lee, J.A., Kaang, B.K., 2003. Synaptic facilitation by ectopic
octopamine and 5-HT receptors in Aplysia. Brain Res. Bull. 60, 73 – 79.
Chen, L., Hamaguchi, K., Hamada, S., Okado, N., 1997. Regional differences of
serotonin-mediated synaptic plasticity in the chicken spinal cord with development
and aging. J. Neural Transplant Plast. 6, 41 – 48.
Chugani, D.C., 2002. Role of altered brain serotonin mechanisms in autism. Mol.
Psychiatry. 7 Suppl 2, S16 – 17.
Colado, M.I., O’Shea, E., Granados, R., Misra, A., Murray, T.K., Green, A.R., 1997.
A study of the neurotoxic effect of MDMA (decstasyT) on 5-HT neurones in the
brains of mothers and neonates following administration of the drug during
pregnancy. Br. J. Pharmacol. 121, 827– 833.
139
Colas, J.F., Launay, J.M., Kellermann, O., Rosay, P., Maroteaux, L., 1994.
Drosophila 5-HT2 serotonin receptor: coexpression with fushi-tarazu during
segmentation. Proc. Natl. Acad. Sci. U.S.A. 92, 5441 – 5445.
Cooper, R.L., Chase, R.J., Tabor, J., 2001. Altered responsiveness to 5-HT at the
crayfish neuromuscular junction due to chronic p-CPA and m-CPP treatment. Brain
Res. 916, 143 – 151.
Cooper, R.L., Donmezer, A., Shearer, J., 2003. Intrinsic differences in sensitivity to
5-HT between high- and low-output terminals innervating the same target.
Neurosci. Res. 45, 163 – 172.
Dailly, E., Chenu, F., Petit-Demouliere, B., Bourin, M., 2006. Specificity and efficacy
of noradrenaline, serotonin depletion in discrete brain areas of Swiss mice by
neurotoxins. J. Neurosci. Methods. 150, 111 – 115.
Djokaj, S., Cooper, R.L., Rathmayer, W., 2001. Effects of octopamine, serotonin,
and cocktails of the two modulators on synaptic transmission at crustacean
neuromuscular junctions. J. Comp. Physiol. A. 187, 145 – 154.
Dourish, C.T., Hutson, P.H., Curzon, G., 1986. Para-chlorophenylalanine prevents
feeding induced by the serotonin agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-
OH-DPAT). Psychopharmacol. 89, 467 – 471.
140
Drummond, P.D., 2006. Tryptophan depletion increases nausea, headache and
photophobia in migraine sufferers. Cephalalgia. 26, 1225 – 1233.
Dulcis, D., Levine, R.B., 2003. Innervation of the heart of the adult fruit fly,
Drosophila melanogaster. J. Comp. Neurol. 465, 560 – 578.
Dulcis, D., Levine, R.B., 2005. Glutamatergic innervation of the heart initiates
retrograde contractions in adult Drosophila melanogaster. J. Neurosci. 25, 271 –
280.
Dulcis, D., Levine, R.B., Ewer, J., 2005. Role of the neuropeptide CCAP in
Drosophila cardiac function. J. Neurobiol. 64, 259 – 274.
Eide, P.K., Hole, K., Berge, O.G., Broch, O.J., 1988. 5-HT depletion with 5,7-DHT,
PCA and PCPA in mice: differential effects on the sensitivity to 5-MeODMT, 8-OH-
DPAT and 5-HTP as measured by two nociceptive tests. Brain Res. 440, 42 – 52.
Encinas, J.M., Vaahtokari, A., Enikolopov, G., 2006. Fluoxetine targets early
progenitor cells in the adult brain. Proc. Natl. Acad. Sci. U. S. A. 103, 8233 – 8238.
Escamilla-Chimal, E.G., Hiriart, M., Sanchez-Soto, M.C., Fanjul-Moles, M.L., 2002.
Serotonin modulation of CHH secretion by isolated cells of the crayfish retina and
optic lobe. Gen. Comp. Endocrinol. 125, 283 – 290.
141
Fickbohm, D.J., Spitzer, N., Katz, P.S., 2005. Pharmacological manipulation of
serotonin levels in the nervous system of the opisthobranch mollusc Tritonia
diomedea. Biol. Bull. 209, 67 – 74.
Filip, M., Frankowska, M., Zaniewska, M., Golda, A., Przegalinski, E., 2005. The
serotonergic system and its role in cocaine addiction. Pharmacol. Rep. 57, 685 –
700.
Filla, A., Hiripi, L., Elekes, K., 2004. Serotonergic and dopaminergic influence of the
duration of embryogenesis and intracapsular locomotion of Lymnaea stagnalis L.
Acta Biol. Hung. 55, 315 – 321.
Fox, L.E., Soll, D.R., Wu, C.F., 2006. Coordination and modulation of locomotion
pattern generators in Drosophila larvae: effects of altered biogenic amine levels by
the tyramine beta hydroxlyase mutation. J. Neurosci. 26, 1486 – 1498.
Gaddum, J.H., Picacelli, Z.P., 1975. Two kinds of tryptamine receptor.
Br. J. Pharmacol. Chemother. 12, 323 – 328.
Gage, F.H., Coates, P.W., Palmer, T.D., Kuhn, H.G., Fisher, L.J., Suhonen, J.O.,
Peterson, D.A., Suhr S.T., Ray J., 1995. Survival and differentiation of adult
neuronal progenitor cells transplanted to the adult brain. Proc. Natl. Acad. Sci. U. S.
A. 92, 11879 – 11883.
142
Garen, A., Kauvar, L., Lepesant, J.A. 1977. Roles of ecdysone in Drosophila
development. Proc. Natl. Acad. Sci. U.S.A. 74, 5099-5103.
Gaspar, P., Cases, O., Maroteaux, L., 2003. The developmental role of serotonin:
news from mouse molecular genetics. Nat. Rev. Neurosci. 4, 1002 – 1012.
Gedye, A., 1990. Dietary increases in serotonin reduces self-injurious behavior in a
Down’s Syndrome adult. J. Ment. Defic. Res. 34, 195 – 203.
Gedye, A., 1991. Serotonergic treatment for aggression in a Down’s Syndrome adult
showing signs of Alzheimer’s Disease. J. Ment. Defic. Res. 35, 247 – 258.
Goldman, S.A., Nottebohm, F., 1983. Neuronal production, migration, and
differentiation in a vocal control nucleus of the adult female canary brain. Proc. Natl.
Acad. Sci. U. S. A. 80, 2390 – 2394.
Gould, E., 1999. Serotonin and hippocampal neurogenesis. Neuropsychopharmacol.
21 46S – 51S.
Green, A.R., Mechan, A.O., Elliott, J.M., O'Shea, E., Colado, M.I., 2003. The
pharmacology and clinical pharmacology of 3,4-methylenedioxymethamphetamine
(MDMA, "ecstasy"). Pharmacol. Rev. 55, 463 – 508.
143
Gresch, P.J., Smith, R.L., Barrett, R.J., Sanders-Bush, E., 2005. Behavioral
tolerance to lysergic acid diethylamide is associated with reduced serotonin-2A
receptor signaling in rat cortex. Neuropsychopharmacol. 30, 1693 –1702.
Gulesserian, T., Engidawork, E., Cairns, N., Lubec, G., 2002. Increased protein
levels of serotonin transporter in frontal cortex of patients with Down syndrome.
Neurosci. Lett. 296, 53 – 57.
Haga, N., Mizumoto, A., Satoh, M., Mochiki, E., Mizusawa, F., Ohshima, K., Itoh, Z.,
1996. Role of endogenous 5-hydroxytryptamine in the regulation of gastric
contractions by motilin in dogs. Am. J. Physiol. 270, G20 – G28.
Hagg, T., 2005. Molecular regulation of adult CNS neurogenesis: an integrated view.
Trends Neurosci. 28, 589 – 595.
Handdwerger, S., Plonk, J.W., Lebowitz, H.E., Biwens, C.H., Feldman, J.M., 1975.
Failure of 5-hydroxytryptophan to stimulate prolactin and growth hormone secretion
in man. J. Clin. Endocrinol. Metab. 45, 588 – 592.
Hanley, H.G., Stahl, S.M., Freedman, D.X., 1977. Hyperserotonemia and amine
metabolites in autistic and retarded children. Arch. Gen. Psychiatry. 34, 521 – 531.
144
Hanley, N.R., Hensler, J.G., 2002. Mechanisms of ligand-induced desensitization of
the 5-hydroxytryptamine (2A) receptor. J. Pharmacol. Exp. Ther. 300, 468-77.
Harzsch, S., Miller, J., Benton, J., Beltz, B., 1999. From embryo to adult: persistent
neurogenesis and apoptotic cell death shape the lobster deutocerebrum. J.
Neurosci. 19, 3472 – 3485.
Herlenius, E., Lagercrantz, H., 2004. Development of neurotransmitter systems
during critical periods. Exp. Neurol. 190, S8 – S21.
Hollander, E., Phillips, A.T., Yeh, C.C., 2003. Targeted treatments for symptom
domains in child and adolescent autism. Lancet. 362, 732 – 734.
Hood, S.D., Hince, D.A., Robinson, H., Cirillo, M., Christmas, D., Kaye, J.M., 2006.
Serotonin regulation of the human stress response. Psychoneuroendocrinol. 31,
1087 – 1097.
Hoyer, D., Hannon, J.P., Martin, G.R., 2002. Molecular, pharmacological and
functional diversity of 5-HT receptors. Pharmacol. Biochem. Behav. 71, 533 – 554.
Huang, G.J., Herbert, J., 2006. Stimulation of neurogenesis in the hippocampus of
the adult rat by fluoxetine requires rhythmic change in corticosterone. Biol.
Psychiatry. 59, 619 – 624.
145
Hubel, D.H., Wiesel, T.N., 1963a. Receptive fields of cells in striate cortex of very
young, visually inexperienced kittens. J. Neurophysiol. 26, 994-1002.
Hubel, D.H., Wiesel, T.N., 1963b. Shape and arrangement of columns in cat striate
cortex. J. Physiol. 165, 559-568.
Insel, T.R., Young, L.J., 2001. The neurobiology of attachment. Nat. Rev. Neurosci.
2, 129 – 136.
Iyengar, B.G., Chou, C.J., Sharma, A., Atwood, H.L., 2006. Modular neuropile
organization in the Drosophila larval brain facilitates identification and mapping of
central neurons. J. Comp. Neurol. 499 (4), 583 – 602.
Jha, S., Rajendran, R., Davda, J., Vaidya, V.A., 2006. Selective serotonin depletion
does not regulate hippocampal neurogenesis in the adult rat brain: differential effects
of p-chlorophenylalanine and 5,7-dihydroxytryptamine. Brain Res. 1075, 48 – 59.
Johnson, E., Ringo, J., Dowse, H., 1997. Modulation of Drosophila heartbeat by
neurotransmitters. J. Comp. Physiol. B. 167, 89 – 97.
146
Johnson, E., Sherry, T., Ringo, J., Dowse, H., 2002. Modulation of the cardiac
pacemaker of Drosophila: cellular mechanisms. J. Comp. Physiol. B. 172, 227 –
236.
Johnstone, A.F., Cooper, R.L., 2006. Direct innervation of the Drosophila
melanogaster larval aorta. Brain Res. 1083, 159 – 163.
Kamyshev, N.G., Smirnova, G.P., Savvateeva, E.V., Medvedeva, A.V.,
Ponomarenko, V.V., 1983. The influence of serotonin and p-chlorophenylalanine on
locomotor activity of Drosophila melanogaster. Pharmacol. Biochem. Behav. 18, 677
– 681.
Khozhai, L.I., Otellin, V.A., 2006. Formation of the neocortex in mice developing in
conditions of prenatal serotonin deficiency. Neurosci. Behav. Physiol. 36, 513 – 517.
Koe, B.K., Weissman, A., 1966. p-Chlorophenylalanine: a specific depletor of brain
serotonin. J. Pharmacol. Exp. Ther. 154, 499 – 516.
Koprich, J.B., Chen, E.Y., Kanaan, N.M., Campbell, N.G., Kordower, J.H., Lipton,
J.W., 2003. Prenatal 3,4-methylenedioxymethamphetamine (ecstasy) alters
exploratory behavior, reduces monoamine metabolism, and increases forebrain
tyrosine hydroxylase fiber density of juvenile rats. Neurotoxicol. Teratol. 25, 509 –
517.
147
Kotak, V.C., Sanes, D.H., 1995.Synaptically evoked prolonged depolarizations in the
developing auditory system. J. Neurophysiol. 74, 1611 – 1620.
Kraft, R., Levine, R.B., Restifo, L.L., 1998. The steroid hormone
20-hydroxyecdysone enhances neurite growth of Drosophila mushroom body
neurons isolated during metamorphosis. J. Neurosci. 18, 8886-8899.
Krzysztof, M.K., 2003. Anesthetic implications of drug abuse in pregnancy, J. Clini.
Anes. 15, 382-394.
Lee, C.Y., Yang, P.F., Zou, H.S., 2001. Serotonergic regulation of crustacean
hyperglycemic hormone secretion in the crayfish, Procambarus clarkii. Physiol.
Biochem. Zool. 74, 376 – 382.
Lee, C.Y., Yau, S.M., Liau, C.S., Huang, W.J., 2000. Serotonergic regulation of
blood glucose levels in the crayfish, Procambarus clarkii: site of action and receptor
characterization. J. Exp. Zool. 286, 596 – 605.
LeVay, S., Wiesel, T.N., Hubel, D.H., 1980. The development of ocular dominance
columns in normal and visually deprived monkeys. J. Comp. Neurol. 191, 1 – 51.
Li, H., Cooper, R.L., 2001. Effects of the ecdysoneless mutant on synaptic efficacy
and structure at the neuromuscular junction in Drosophila larvae during normal and
prolonged development. Neurosci. 106,193-200.
148
Li, H., Harrison, D., Jones, G., Jones, D., Cooper, R.L., 2001. Alterations in
development, behavior, and physiology in Drosophila larva that have reduced
ecdysone production. J. Neurophysiol. 85, 98-104.
Lie, D.C., Song, H., Colamarino, S.A., Ming, G.L., Gage, F.H., 2004. Neurogenesis
in the adult brain: new strategies for central nervous system diseases. Annu. Rev.
Pharmacol. Toxicol. 44, 399 – 421.
Lippe, W.R., 1994. Rhythmic spontaneous activity in the developing avian auditory
system. J. Neurosci. 14, 1486 – 1495.
Listerman, L.R., Deskins, J., Bradacs, H., Cooper, R.L., 2000. Heart rate within male
crayfish: social interactions and effects of 5-HT. Comp. Biochem. Physiol. A Mol.
Integr. Physiol. 125, 251 – 263.
Livingston, M.S., Harris-Warrick, R.M., Kravitz, E.A., 1980. Serotonin and
octopamine produce opposite postures in lobsters. Science 208, 76 – 79.
Lois, C., Alvarez-Buylla, A., 1993. Proliferating subventricular zone cells in the adult
mammalian forebrain can differentiate into neurons and glia. Proc. Natl. Acad. Sci.
U. S. A. 90, 2074 – 2077.
149
Luskin, M.B., 1993. Restricted proliferation and migration of postnatally generated
neurons derived from the forebrain subventricular zone. Neuron. 11, 173 – 189.
Lyles, J., Cadet, J.L., 2003. Methylenedioxymethamphetamine (MDMA, Ecstasy)
neurotoxicity: cellular and molecular mechanisms. Brain Res. Brain Res. Rev. 42,
155 –168.
Maggi, L., Le Magueresse, C., Changeux, J.-P., Cherubini, E., 2003. Nicotine
activates immature ‘‘silent’’ connections in the developing hippocampus. Proc. Natl.
Acad. Sci. U.S.A. 18, 2059 – 2064.
Mann, D. M., Yates, P. O., Marcyniuk, B., Ravindra, C. R., 1985. Pathological
deficits in Down’s Syndrome of middle age. J. Ment. Defic. Res. 29, 125 – 135.
Marinesco, S., Carew, T.J., 2002. Serotonin release evoked by tail nerve stimulation
in the CNS of aplysia: characterization and relationship to heterosynaptic plasticity.
J. Neurosci. 22, 2299 – 2312.
Marinesco, S., Kolkman K.E., Carew, T.J., 2004. Serotonergic modulation in aplysia.
I. Distributed serotonergic network persistently activated by sensitizing stimuli. J.
Neurophysiol. 92, 2468 – 2486.
150
Mattson, M.P., Spaziani, E., 1986. Regulation of the stress-responsive X-organ--Y-
organ axis by 5-hydroxytryptamine in the crab, Cancer antennarius. Gen. Comp.
Endocrinol. 62, 419 – 427.
McDonald, J.W., Liu, X.Z., Qu, Y., Liu, S., Mickey, S.K., Turetsky, D., Gottlieb D.I.,
Choi, D.W., 1999. Transplanted embryonic stem cells survive, differentiate and
promote recovery in injured rat spinal cord. Nat. Med. 5, 1410 – 1412.
Meltzer, H.Y., Li, Z., Kaneda, Y., Ichikawa, J., 2003. Serotonin receptors: their key
role in drugs to treat schizophrenia. Prog. Neuropsychopharmacol. Biol. Psychiatry.
27, 1159 – 1172.
Miller, R.H., 2006. The promise of stem cells for neural repair.
Brain Res. 1091, 258 – 264.
Mnie-Filali, O., El-Mansari, M., Espana, A., Sanchez, C., Haddjeri, N., 2006.
Allosteric modulation of the effects of the 5-HT reuptake inhibitor escitalopram on the
rat hippocampal synaptic plasticity. Neurosci. Lett. 395, 23 –27.
Monastirioti, M., 1999. Biogenic amine systems in the fruit fly Drosophila
melanogaster. Microsc. Res. Tech. 45, 106 – 121.
151
Monfils, M.H., Driscoll, I., Kamitakahara, H., Wilson, B., Flynn, C., Teskey, G.C.,
Kleim, J.A., Kolb, B., 2006. FGF-2-induced cell proliferation stimulates anatomical,
neurophysiological and functional recovery from neonatal motor cortex injury. Eur. J.
Neurosci. 24, 739 – 749.
Mota, A., Bento, A., Penalva, A., Pombo, M., Dieguez, C. 1995. Role of the
serotonin receptor subtype 5-HT1D on basal and stimulated growth hormone
secretion. J. Clin .Endocrinol. Metab. 80, 1973 – 1977.
Murakami, Y., Kato, Y., Kabayama, Y., Tojo, K., Inoue, T., Imura, H., 1986.
Involvement of growth hormone-releasing factor in GH secretion induced by
serotoninergic mechanisms in conscious rats. Endocrinol. 119, 1089 – 1092.
Namerow, L.B., Thomas, P., Bostic, J.Q., Prince, J., Monuteaux, M.C., 2003. Use of
citalopram in pervasive developmental disorders. J. Dev. Behav. Pediatr. 24, 104 –
108.
Naqui, S.Z.H., Harris, B.S., Thomaidou, D., Parnavelas, J.G., 1999. The
noradrenergic system influences in fate of Cajal-Retzius cells in the developing
cerebral cortex. Dev. Brain Res. 113, 75 – 82.
152
Nichols, C.D., Ronesi, J., Pratt, W., Sanders-Bush, E., 2002. Hallucinogens and
Drosophila: linking serotonin receptor activation to behavior. Neurosci. 115, 979 –
984.
Nichols, D.E., 2004. Hallucinogens. Pharmacol. Ther. 101, 131 – 181.
Nichols, R., McCormick, J., Cohen, M., Howe, E., Jean, C., Paisley, K., Rosario, C.,
1999. Differential processing of neuropeptides influences Drosophila heart rate. J.
Neurogenet. 13, 89 -104.
Niswender, C.M., Sanders-Bush, E., Emeson, R.B., 1998. Identification and
characterization of RNA editing events within the 5-HT2C receptor. Ann. N Y Acad.
Sci. 861, 38 – 48.
Nottebohm, F., 1980. Testosterone triggers growth of brain vocal control nuclei in
adult female canaries. Brain Res. 189, 429 – 436.
Nottebohm, F., Arnold, A.P., 1976. Sexual dimorphism in vocal control areas of the
songbird brain. Science. 194, 211 – 213.
Nottebohm, F., Nottebohm, M. E., Crane, L., 1986. Developmental and seasonal
changes in canary song and their relation to changes in the anatomy of song-control
nuclei. Behav. Neural Biol. 46, 445 – 471.
153
O'Donovan, M., Ho, S., Yee, W., 1994. Calcium imaging of rhythmic network activity
in the developing spinal cord of the chick embryo. J. Neurosci. 14, 6354 –6369.
Olson, L., Seiger, A., 1972. Early prenatal ontogeny of central monoamine neurons
in the rat: fluorescence histochemical observations. Z. Anat. Entwicklungsgesch.
137, 301 – 316.
Osborne, R. H., 1996. Insect Neurotransmission: Neurotransmitters and Their
Receptors. Pharmacol. Ther. 69, 117-142.
Pak, M.D., Gilbert, L.I., 1987. A developmental analysis of ecdysteroids during the
metamorphosis of Drosophila melanogaster. J. Liq. Chrom. 10, 2591-2611.
Papaefthmiou, C., Theophilidis, G., 2001. An in vitro method for recording the
electrical activity of the isolated heart of the adult Drosophila melanogaster.
In Vitro Cell Dev. Biol. Anim. 37, 445 – 449.
Pellegrino, T.C., Bayer, B.M., 2000. Specific serotonin reuptake inhibitor-induced
decreases in lymphocyte activity require endogenous serotonin release.
Neuroimmunomodula. 8, 179 – 187.
154
Pendleton, R.G., Parvez, F., Sayed, M., Hillman, R., 2002. Effects of
pharmacological agents upon a transgenic model of Parkinson's disease in
Drosophila melanogaster. J. Pharmacol. Exp. Ther. 300, 91 – 96.
Penn, A.A., Shatz, C.J., 1999. Brain waves and brain wiring: the role of endogenous
and sensory-driven neural activity in development. Pediatr. Res. 45, 447 – 458.
Peroutka, S.J., 1994. Molecular biology of serotonin (5-HT) receptors. Synapse. 18,
241 – 260.
Purves, D., Lichtman, J.W., 1987. Synaptic sites on reinnervated nerve cells
visualized at two different times in living mice. J. Neurosci. 7, 1492 – 1497.
Rapport, M.M., Green, A.A., Page, I.H., 1948a. Partial purification of the
vasoconstrictor in beef serum. J. Biol. Chem. 174, 735 – 738.
Rapport, M.M., Green, A.A., Page, I.H., 1948b. Crystalline Serotonin. Science 108,
329 – 330.
Reissig, C.J., Eckler, J.R., Rabin, R.A., Winter, J.C., 2005. The 5-HT1A receptor and
the stimulus effects of LSD in the rat. Psychopharmacol (Berl). 182, 197 – 204.
155
Reynolds, G.P., Templeman, L.A., Zhang, Z.J., 2005. The role of 5-HT2C receptor
polymorphisms in the pharmacogenetics of antipsychotic drug treatment. Prog
Neuropsychopharmacol. Biol. Psychiatry. 29, 1021 – 1028.
Roeder, T., 1999. Octopamine in invertebrates. Prog. Neurobiol. 59, 533 - 561.
Roth, B.L., Willins, D.L., Kristiansen, K., Kroeze, W.K., 1998. 5-Hydroxytryptamine2-
family receptors (5-hydroxytryptamine2A, 5-hydroxytryptamine2B, 5-
hydroxytryptamine2C): where structure meets function. Pharmacol. Ther. 79, 231 –
2 57.
Rothenfluh, A., Heberlein, U., 2002. Drugs, flies, and videotape: the effects of
ethanol and cocaine on Drosophila locomotion. Curr. Opin. Neurobiol. 12, 639 – 645.
Sanders-Bush, E., Massari, V.J., 1977. Actions of drugs that deplete serotonin. Fed.
Proc. 36, 2149 – 2153.
Sanes, J.R., Lichtman, J.W., 1999. Development of the vertebrate neuromuscular
junction. Annu. Rev. Neurosci. 22, 389 – 442.
Saudou, F., Boschert, U., Amlaiky, N., Plassat, J.L., Hen, R., 1992. A family of
Drosophila serotonin receptors with distinct intracellular signaling properties and
expression patterns. J. EMBO. 11, 7-17.
156
Saudou, F., Hen, R., 1994. 5-Hydroxytryptamine receptor subtypes in vertebrates
and invertebrates. Neurochem. Int. 25, 503 – 532.
Schulz, D.J., Barron, A.B., Robinson, G.E., 2002. A role for octopamine in honey
bee division of labor. Brain Behav. Evol. 60, 350 – 359.
Sherman, S.M., Spear, P.D., 1982. Organization of visual pathways in normal and
visually deprived cats. Physiol. Rev. 62, 738 – 855.
Shirahata, T., Tsunoda, M., Santa, T., Kirino, Y., Watanabe, S., 2006. Depletion of
serotonin selectively impairs short-term memory without affecting long-term memory
in odor learning in the terrestrial slug Limax valentianus. Learn Mem. 13, 267 – 270.
Shuranova, Z.P., Burmistrov, Y.M., Strawn, J.R., Cooper, R.L., 2006. Evidence for
an Autonomic Nervous System in Decapod Crustaceans. Int. J. of Zool. Res. 2, 242
– 283.
Simantov, R., 2004. Multiple molecular and neuropharmacological effects of MDMA
(Ecstasy). Life Sci. 74, 803 – 814.
Sinha, R.K. 2006. P-CPA pretreatment reverses the changes in sleep and behavior
following acute immobilization stress rats. J. Physiol. Sci. 56, 123 - 129.
157
Sodhi, M.S., Sanders-Bush, E., 2004. Serotonin and brain development. Int. Rev.
Neurobiol. 59, 111 – 174.
Stimmel, G.L., Gutierrez, M.A., Lee, V., 2002. Ziprasidone: an atypical antipsychotic
drug for the treatment of schizophrenia. Clin. Ther. 24, 21 – 37.
Stryker, M.P., Harris, W.A., 1986. Binocular impulse blockade prevents the formation
of ocular dominance columns in cat visual cortex. J. Neurosci. 6, 2117 – 2133.
Sundstrom, E., Kolare, S., Souverbie, F., Samuelsson, E.B., Pschera, H., Lunell,
N.O., Seiger, A., 1993. Neurochemical differentiation of human bulbospinal
monoaminergic neurons during the first trimester. Brain Res. Dev. Brain Res. 75, 1 –
12.
Thummel, C.S., 1996. Flies on steroids-Drosophila metamorphosis and the
mechanisms of steroid hormone action. Trends in Genetics. 12, 306 – 310.
Tierney, A.J., 2001. Structure and function of invertebrate 5-HT receptors: a review.
Comp. Biochem. Physiol. A Mol. Integr. Physiol. 128, 791 – 804.
Truman, J.W., 1996. Steroid receptors and nervous system metamorphosis in
insects. Dev. Neurosci. 18, 87-101.
158
Truman, J.W., Reiss, S.W., 1988. Hormonal regulation of the shape of identified
motoneurons in the moth Manduca Sexta. J. Neurosci. 8, 763 - 775.
Twarog, B.M., Page, I.H., 1953. Serotonin content of some mammalian tissues and
urine and a method for its determination. Am. J. Physiol. 175, 157 – 161
Vaysse, G., Galissie, M., Corbiere, M., 1988. Induced variation of serotonin in
Drosophila melanogaster and its relation to learning performance. J. Comp. Psychol.
102, 225 – 229.
Verhage, M., Maia, A.S., Plomp, J.J., Brussaard, A.B., Heeroma, J.H.,Vermeer, H.,
Toonen, R.F., Hammer, R.E., van den Berg, T.K., Missler,M., Geuze, H.J., Sudhof,
T.C., 2000. Synaptic assembly of the brain in the absence of neurotransmitter
secretion. Science 287, 864– 869.
Walker, S.C., Mikheenko, Y.P., Argyle, L.D., Robbins, T.W., Roberts, A.C., 2006.
Selective prefrontal serotonin depletion impairs acquisition of a detour-reaching task.
Eur. J. Neurosci. 23, 3119 – 3123.
Warren, R.P., Singh, V.K., 1996. Elevated serotonin levels in autism: association
with the major histocompatibility complex. Neuropsychobiol. 34, 72 – 75.
Whitaker-Azmitia, P.M., 1998. Role of the neurotrophic properties of serotonin in the
delay of brain maturation induced by cocaine. Ann. N. Y. Acad. Sci. 846, 158 – 164.
159
Whitaker-Azmitia, P.M., 1999. The discovery of serotonin and its role in
neuroscience. Neuropsychopharmacol. 21 (2 Suppl), 2S-8S.
Whitaker-Azmitia, P.M., 2001. Serotonin and brain development: role in human
developmental diseases. Brain Res. Bull. 56, 479 – 485.
Wiesel, T.N., 1982. Postnatal development of the visual cortex and the influence of
environment. Nature. 299, 583 – 591.
Wiesel, T.N., Hubel, D.H., 1965. Extent of recovery from the effects of visual
deprivation in kittens. J. Neurophysiol. 28, 1060 – 1072.
Willard, S.S., Koss, C.M., Cronmiller, C., 2006. Chronic cocaine exposure in
Drosophila: life, cell death and oogenesis. Dev. Biol. 296, 150 – 163.
Witz, P.,1990. Cloning and chacterization of a Drosophila serotonin receptor that
activates adenylate cyclase. Neurobiol. 87, 8940-8944.
Woolley, D.W., Shaw, E., 1954. A biochemical and pharmacological suggestion
about certain mental disorders. Proc. Natl. Acad. Sci. U.S.A. 40, 228 – 231
160
Woolverton, W.L., Johnson, K.M., 1992. Neurobiology of cocaine abuse. Trends
Pharmacol. Sci. 13, 193 – 200.
Yan, W., Wilson, C.C., Haring, J.H., 1997. 5-HT1a receptors mediate the
neurotrophic effect of serotonin on developing dentate granule cells. Brain Res. Dev.
Brain. Res. 98, 185 – 190.
Zhang Li. I., Poo M.M., 2001. Electrical activity and development of neural circuits.
Nat. Neurosci. 4 Suppl, 1207 – 1214.
Zhou, J.F., Chen, P., Zhou, Y.H., Zhang, L., Chen, H.H., 2003. 3,4-
Methylenedioxymethamphetamine (MDMA) abuse may cause oxidative stress and
potential free radical damage. Free Radic. Res. 37, 491 – 497.
Zhou, Q.Y., Palmiter, R.D., 1995a. Dopamine-deficient mice are severely
hypoactive, adipsic, and aphagic. Cell 83, 1197 – 1209.
Zhou, Q.Y., Quaife, C.J., Palmiter, R.D., 1995b. Targeted disruption of the tyrosine
hydroxylase gene reveals that catecholamines are required for mouse fetal
development. Nature 374, 640 – 643.
Zornik, E., Paisley, K., Nichols, R., 1999. Neural transmitters and a peptide modulate
Drosophila heart rate. Peptides. 20, 45 – 51.
161
CHAPTER 2 Atwood, H.L., Govind, C.K., Wu, C.F., 1993. Differential ultrastructure of synaptic
terminals on ventral longitudinal abdominal muscles in Drosophila larvae. J.
Neurobio. 24, 1008 – 1024.
Baier, A., Wittek, B., Brembs, B., 2002. Drosophila as a new model organism for the
neurobiology of aggression? J. Exp. Biol. 205, 1233 – 1240.
Ball, R., Xing, B., Bonner, P., Shearer, J., Cooper, R.L., 2003. Long-term
maintenance of neuromuscular junction activity in cultured Drosophila larvae. Comp.
Biochem. Physiol. A. 134, 247 – 255.
Blenau, W., Baumann, A., 2001. Molecular and pharmacological properties of insect
biogenic amine receptors: Lessons from Drosophila melanogaster and Apis
mellifera. Arch. Insect Biochem. Physiol. 48, 13 – 38.
Campos-Ortega, J.A., Hartenstein, V. (Eds.), In: The embryonic development of
Drosophila melanogaster. Spring-Verlag, Berlin, 1985.
162
Chau, C., Giroux, N., Barbeau, H., Jordan, L., Rossignol, S., 2002. Effects of
intrathecal glutamatergic drugs on locomotion I. NMDA in short-term spinal cats. J.
Neurophysiol. 88, 3032 – 3045.
Cooper, R.L., Govind, C.K. ,1991. Axon composition of the proprioceptive PD nerve
during growth and regeneration of lobster claws. J. Exp. Zool. 260, 181-193.
Cooper, R.L., Neckameyer, W.S., 1999. Dopaminergic neuromodulation of motor
neuron activity and neuromuscular function in Drosophila melanogaster. Comp.
Biochem. Physiol. B. 122, 199 – 210.
Cooper, R.L., Stewart, B.A., Wojtowicz, J.M., Wang, S., Atwood, H.L., 1995. Quantal
measurement and analysis methods compared for crayfish and Drosophila
neuromuscular junctions and rat hippocampus. J. Neurosci. Meth. 61, 67 – 78.
Djokaj, S., Cooper, R.L., Rathmayer, W., 2001. Effects of octopamine, serotonin,
and cocktails of the two modulators on synaptic transmission at crustacean
neuromuscular junctions. J. Comp. Physiol. A. 187, 145 – 154.
Francis, M.M., Mellem, J.E., Maricq, A.V., 2003. Bridging the gap between genes
and behaviour: Recent advances in electrophysiological analysis of neural function
in Caenorhabditis elegans. Trends in Neurosci. 26, 90 – 99.
163
Friggi-Grelin, F., Coulom, H., Meller, M.,Gomez, D., Hirsh, J., Birman, S., 2003.
Targeted gene expression in Drosophila dopaminergic cells using regulatory
sequences from tyrosine hydroxylase. J. Neurobiol. 54, 618 – 627.
Garen, A., Kauvar, L., Lepesant, J.A. 1977. Roles of ecdysone in Drosophila
development. Proc. Natl. Acad. Sci. U. S. A. 74, 5099 – 5103.
Govind, C.K., Pearce, J., 1985. Lateralization in number and size of sensory axons
to the dimorphic chelipeds of crustaceans. J. Neurobiol. 16, 111 – 125.
Govind, C.K., Pearce, J., 1986. Differential reflex activity determines claw and
closer muscle asymmetry in developing lobsters. Science 233, 354 – 356.
Govind, C.K., Pearce, J., Potter, D.J., 1988. Neural attrition following limb loss and
regeneration in juvenile lobsters. J. Neurobiol. 19, 667 – 680.
Han, K.A., Millar, N.S., Davis, R.L., 1998. A novel octopamine receptor with
preferential expression in Drosophila mushroom bodies. J. Neurosci. 18, 3650 –
3658.
Hirashima, A., Sukhanova, M.Jh., Rauschenbach, I.Yu., 2000. Genetic control of
biogenic-amine systems in Drosophila under normal and stress conditions. Biochem.
Genet. 38, 167 – 180.
164
Hubel, D.H., Wiesel, T.N., 1963a. Receptive fields of cells in striate cortex of very
young, visually inexperienced kittens. J. Neurophysiol. 26, 994 – 1002.
Hubel, D.H., Wiesel, T.N., 1963b. Shape and arrangement of columns in cat striate
cortex. J. Physiol. 165, 559 – 568.
Hubel, D.H., Wiesel, T.N., 1968. Receptive fields and functional architecture of
monkey striate cortex. J. Physiol. 195, 215 – 243.
Hubel, D.H., Wiesel, T.N., 1970. The period of susceptibility to the physiological
effects of unilateral eye closure in kittens. J. Physiol. 206, 419 – 436.
Johnson, E., Ringo, J., Dowse, H., 1997. Modulation of Drosophila heart beat by
neurotransmitters. J. Comp. Physiol. B. 167, 89 – 97.
Kraft, R., Levine, R.B., Restifo, L.L., 1998. The steroid hormone
20-hydroxyecdysone enhances neurite growth of Drosophila mushroom body
neurons isolated during metamorphosis. J. Neurosci. 18, 8886 – 8899.
Kurdyak, P., Atwood, H.L., Stewart, B.A., Wu, C.F., 1994. Differential physiology
and morphology of motor axons to ventral longitudinal muscle in larval Drosophila. J.
Comp. Neurol. 350, 463 – 472.
165
Lang, F., Govind, C.K., Costello, W.J., 1978. Experimental transformation of muscle
fiber properties in lobster. Science 201,1037 – 1039.
Li, H., Cooper, R.L., 2001. Effects of the ecdysoneless mutant on synaptic efficacy
and structure at the neuromuscular junction in Drosophila larvae during normal and
prolonged development. Neurosci. 106,193 – 200.
Li, H., Harrison, D., Jones, G., Jones, D., Cooper, R.L., 2001. Alterations in
development, behavior, and physiology in Drosophila larva that have reduced
ecdysone production. J. Neurophysiol. 85, 98 – 104.
Li, H., Peng, X., Cooper, R.L., 2002. Development of Drosophila larval
neuromuscular junctions: Maintaining synaptic strength. Neurosci. 115, 505 – 513.
Macleod, G.T., Hegström-Wojtowicz, M., Charloton, M.P., Atwood, H.L., 2002. Fast
calcium signals in Drosophila motor neuron terminals. J. Neurophysiol. 88, 2659 –
2663.
Marder, E., Thirumalai, V., 2002, Cellular, synaptic and network effects of
neuromodulation. Neural Networks 15, 479 – 493.
Monastirioti, M., 1999. Biogenic amine systems in the fruit fly Drosophila
melanogaster. Microscopy Res. Tech. 45, 106 – 121.
166
Neckameyer, W.S., 1998a. Dopamine and mushroom bodies in Drosophila
melanogaster: experience-dependent and -independent aspects of sexual behavior.
Lear. Mem. 5, 157 – 165.
Neckameyer, W.S., 1998b. Dopamine modulates female sexual receptivity in
Drosophila melanogaster. J. Neurogenet. 12, 101 – 114.
Neckameyer, W.S., Cooper, R.L., 1998. GABA transporters in Drosophila
melanogaster: developmental expression, behavior, and physiology. Invert.
Neurosci. 3, 279 – 294.
Pak, M.D., Gilbert, L.I., 1987. A developmental analysis of ecdysteroids during the
metamorphosis of Drosophila melanogaster. J. Liq. Chrom. 10, 2591 – 2611.
Pallas, S.L., 2001. Intrinsic and extrinsic factors that shape neocortical specification.
Trends Neurosci. 24, 417 – 423.
Rossignol, S., 2000. Locomotion and its recovery after spinal injury. Curr. Opin.
Neurobiol. 10, 708 – 716.
167
Rossignol, S., Bouyer, L., Barthelemy, D., Langlet, C., Leblond, H., 2002. Recovery
of locomotion in the cat following spinal cord lesions. Brain Res. Brain Res. Rev. 40,
257 – 266.
Rossignol, S., Giroux, N., Chau, C., Marcoux, J., Brustein, J., Reader, T.A., 2001.
Pharmacological aids to locomotor training after spinal injury in the cat. J. Physiol.
533, 65 – 74.
Ruffner, M.E., Cromarty, S.I., Cooper, R.L., 1999. Depression of synaptic efficacy in
high- and low-output Drosophila neuromuscular junctions by the molting hormone
(20-Hydroxyecdysone) during intermolt. J. Neurophysiol. 81, 788 – 794.
Sherrington, C.S., 1898. Decerebrate rigidity, and reflex coordination of movements.
J. Physiol. (Lond). 22, 319 – 332.
Stewart, B.A., Atwood, H.L., Renger, J.J., Wang, J., Wu, C.F.,1994. Improved
stability of Drosophila larval neuromuscular preparation in haemolymph-like
physiological solutions. J. Comp. Physiol. A. 175, 179 – 191.
Suster, M.L., Bate, M., 2002. Embryonic assembly of a central pattern generator
without sensory input. Nature 416, 174 – 178.
168
Svensson, E., Woolley, J., Wikström M., Grillner, S., 2003. Endogenous
dopaminergic modulation of the lamprey spinal locomotor network. Brain Res. 970, 1
- 8.
Taylor, D.J, Robinson, G.E., Logan, B.J., Laverty, R., Mercer, A.R., 1992. Changes
in brain amine levels associated with morphological and behavioural development of
the worker honeybee. J. Comp. Physiol. A. 170, 715 – 721.
Thummel, C.S., 1996. Flies on steroids-Drosophila metamorphosis and the
mechanisms of steroid hormone action. Trends in Genetics. 12, 306 – 310.
Truman, J.W., 1996. Steroid receptors and nervous system metamorphosis in
insects. Dev. Neurosci. 18, 87 – 101.
Truman, J.W., Reiss, S.W., 1988. Hormonal regulation of the shape of identified
motoneurons in the moth Manduca Sexta. J. Neurosci. 8, 763 – 775.
Weeks, J.C., 1981. Neuronal basis of leech swimming: Separation of swim initiation,
pattern generation and intersegmental coordination by selective lesions. J.
Neurophysiol. 45, 698 – 723.
Willard, A.L., 1981. Effects of serotonin on the generation of the motor program for
swimming by the medicinal leech. J. Neurosci. 1, 936 – 944.
169
Zornik, E., Paislet, K., Nichols, R., 1999. Neural transmitters and peptide modulate
Drosophila heart rate. Peptides 20, 45 – 51.
CHAPTER 3 Aréchiga, H., Huberman, A., 1980. Hormonal modulation of circadian be-havior in
crustaceans, Front. Horm. Res., 6, 16 – 34.
Aubert, R., Betoulle, D., Herbeth, B., Siest, G., Fumeron, F., 2000. 5-HT2A receptor
gene polymorphism is associated with food and alcohol intake in obese people. Int.
J. Obes. Relat. Metab. Disord. 24, 920 – 924.
Ayali, A., Harris-Warrick, R. M., 1999. Monoamine control of the pacemaker kernel
and cycle frequency in the lobster pyloric network. J. Neurosci. 19, 6712 – 6722.
Barnes, N.M., Sharp, T., 1999. A review of central 5-HT receptors and their function.
Neuropharmacol. 38, 1083 – 1152.
Bicker, G., 1999. Biogenic amines in the brain of the honeybee: cellular distribution,
development, and behavioral functions. Microsc. Res. Tech. 44, 166 – 178.
Blenau, W., Baumann, A., 2001. Molecular and pharmacological properties of insect
biogenic amine receptors: lessons from Drosophila melanogaster and Apis mellifera.
Arch. Insect Biochem. Physiol. 48, 13 – 38.
170
Campos-Ortega, J.A., Hartenstein, V., 1985. In: The embryonic development of
Drosophila melanogaster. Spring-Verlag, Berlin.
Cesani, M.F., Orden, A.B., Oyhenart, E.E., Zucchi, M., Mune, M.C., Pucciarelli,
H.M., 2006. Growth of functional cranial components in rats submitted to
intergenerational undernutrition. J. Anat. 209, 137 – 147.
Chen, B., Meinertzhagen, I.A., Shaw, S.R., 1999. Circadian rhythms in light-evoked
responses of the fly's compound eye, and the effects of neuromodulators 5-HT and
the peptide PDF. J. Comp. Physiol. A. 185, 393 – 404.
Clark, M.C., Dever, T.E., Dever, J.J., Xu, P., Rehder, V., Sosa, M.A., Baro, D.J.,
2004. Arthropod 5-HT2 receptors: a neurohormonal receptor in decapod
crustaceans that displays agonist independent activity resulting from an evolutionary
alteration to the DRY motif. J. Neurosci. 24, 3421 – 3435.
Colas, J.F., Launay, J.M., Maroteaux, L., 1999. Maternal and zygotic control of
serotonin biosynthesis are both necessary for Drosophila germband extension.
Mech Dev. 87, 67 – 76.
Coleman, C.M., Neckameyer, W.S., 2005. Serotonin synthesis by two distinct
enzymes in Drosophila melanogaster. Arch. Insect Biochem. Physiol. 59, 12 – 31.
171
Cooper, R.L., Neckameyer, W.S., 1999. Dopaminergic neuromodulation of motor
neuron activity and neuromuscular function in Drosophila melanogaster. Comp.
Biochem. Physiol. B. 122, 199 – 210.
Cooper, R.L., 1998. Development of sensory processes during limb regeneration in
adult crayfish. J. Exp. Biol. 201, 1745 – 1752.
Cooper, R.L., Li, H., Long, L.Y., Cole, J., Hopper, H.L., 2001. Anatomical
comparisons of neural systems in sighted epigean & troglobitic crayfish species. J.
Crustacean Biol. 21, 360 – 374.
Cooper, R.L., Stewart, B.A., Wojtowicz, J.M., Wang, S., Atwood, H.L., 1995. Quantal
measurement and analysis methods compared for crayfish and Drosophila
neuromuscular junctions and rat hippocampus. J. Neurosci. Meth. 61, 67 – 78.
Cooper, R.L., Winslow, J., Govind, C.K., Atwood, H.L., 1996. Synaptic structural
complexity as a factor enhancing probability of calcium-mediated transmitter release.
J. Neurophysiol. 75, 2451 – 2466.
Copenhaver, P.F., Taghert, P.H., 1989. Development of the enteric nervous system
in the moth. II. Stereotyped cell migration precedes the differentiation of embryonic
neurons. Dev. Biol. 131, 85 – 101.
172
Copenhaver, P.F., Taghert, P.H., 1991. Origins of the insect enteric nervous system:
differentiation of the enteric ganglia from a neurogenic epithelium. Development.
113, 1115 – 1132.
Curtius, H.C., Niederwieser, A., Viscontini, M., Leimbacher, W., Wegmann, H.,
Blehova, B., Rey, F., Schaub, J., Schmidt, H., 1981. Serotonin and dopamine
synthesis in phenylketonuria. Adv. Exp. Med. Biol. 133, 277 – 291.
Dasari, S., Cooper, R.L., 2004. Modulation of sensory to motor circuits by serotonin,
octopamine, and dopamine in semi-intact Drosophila larva. Neurosci. Res. 48, 221 –
227.
Dasari, S., Cooper, R.L., 2005. Influence of the serotonergic system on physiology,
development, and behavior of Drosophila melanogaster. Program No. 176.5.
Dasari, S., Cooper, R.L., 2006. Direct influence of serotonin on the larval heart of
Drosophila melanogaster. J. Comp. Physiol., B. 176, 349 – 357.
De La Torre, R., Farre, M., 2004. Neurotoxicity of MDMA (ecstasy): the limitations of
scaling from animals to humans. Trends Pharmacol. Sci. 25, 505 – 508.
Demchyshyn, L.L., Pristupa, Z.B., Sugamori, K.S., Barker, E.L., Blakely, R.D.,
Wolfgang, W.J., Forte, M.A., Niznik, H.B., 1994. Cloning, expression, and
173
localization of a chloride-facilitated, cocaine-sensitive serotonin transporter from
Drosophila melanogaster. Proc. Natl. Acad. Sci. U. S. A. 91, 5158 – 5162.
Fickbohm, D.J., Katz, P.S., 2000. Paradoxical actions of the serotonin precursor 5-
hydroxytryptophan on the activity of identified serotonergic neurons in a simple
motor circuit. J Neurosci. 20, 1622 – 1634.
Friggi-Grelin, F., Coulom, H., Meller, M., Gomez, D., Hirsh, J., Birman, S., 2003.
Targeted gene expression in Drosophila dopaminergic cells using regulatory
sequences from tyrosine hydroxylase. J. Neurobiol. 54, 618 – 627.
Green, A.R., Mechan, A.O., Elliott, J.M., O'Shea, E., Colado, M.I., 2003. The
pharmacology and clinical pharmacology of 3,4 methylenedioxy-methamphetamine
(MDMA, "ecstasy"). Pharmacol. Rev. 55, 463 – 508.
Hall, M.E., Hoffer, B.J., Gerhardt, G.A., 1989. Rapid and sensitive determination of
catecholamines in small tissue samples by high performance liquid chromatography
coupled with dual-electrode coulometric electrochemical detection. LC-GC. 7, 258
– 275.
Han, K.A., Millar, N.S., Davis, R.L., 1998. A novel octopamine receptor with
preferential expression in Drosophila mushroom bodies. J.Neurosci. 18, 3650 –
3658.
174
Hill, E.S., Okada, K., Kanzaki, R., 2003. Visualization of modulatory effects of
serotonin in the silkmoth antennal lobe. J. Exp. Biol. 206, 345 – 352.
Hirashima, A., Sukhanova, M.Jh., Rauschenbach, I.Yu., 2000. Genetic control of
biogenic-amine systems in Drosophila under normal and stress conditions. Biochem.
Genet. 38, 167 – 180.
Hubel, D.H., Wiesel, T.N., 1963a. Receptive fields of cells in striate cortex of very
young, visually inexperienced kittens. J. Neurophysiol. 26, 994 – 1002.
Hubel, D.H., Wiesel, T.N., 1963b. Shape and arrangement of columns in cat striate
cortex. J. Physiol., 165, 559 – 568.
Hubel, D.H., Wiesel, T.N., 1968. Receptive fields and functional architecture of
monkey striate cortex. J. Physiol. 195, 215 – 243.
Hubel, D.H., Wiesel, T.N., 1970. The period of susceptibility to the physiological
effects of unilateral eye closure in kittens. J. Physiol. 206, 419 – 436.
Johnson, E., Ringo, J., Dowse, H., 1997. Modulation of Drosophila heartbeat by
neurotransmitters. J. Comp. Physiol. B. 167, 89 – 97.
175
Johnson, E., Ringo, J., Dowse, H., 2000. Native and heterologous neuropeptides are
cardioactive in Drosophila melanogaster. J. Insect Physiol. 46, 1229 – 1236.
Johnstone, A.F.M., Cooper, R.L., 2006. Direct innervation of the Drosophila
melanogaster larval heart. Brain Res. 1083, 159 – 163.
Kamyshev, N.G., Smirnova, G.P., Savvateeva, E.V., Medvedeva, A.V.,
Ponomarenko, V.V., 1983. The influence of serotonin and p-chlorophenylalanine on
locomotor activity of Drosophila melanogaster. Pharmacol. Biochem. Behav. 18, 677
– 681.
Katz, P.S., Harris-Warrick, R.M., 1989. Serotonergic/cholinergic muscle receptor
cells in the crab stomatogastric nervous system. II. Rapid nicotinic and prolonged
modulatory effects on neurons in the stomatogastric ganglion. J. Neurophysiol. 62,
571 – 581.
Kishore, S., Stamm, S., 2006. The snoRNA HBII-52 regulates alternative splicing of
the serotonin receptor 5-HT2CR. Science. 311, 230 – 232.
Kloppenburg, P., Hildebrand, J.G., 1995. Neuromodulation by 5-hydroxytryptamine
in the antennal lobe of the sphinx moth Manduca sexta.
J. Exp. Biol. 198, 603 – 611.
176
Krobert, K.A., Levy, F.O., 2002. The human 5-HT7 serotonin receptor splice
variants: constitutive activity and inverse agonist effects.
Br. J. Pharmacol. 135, 1563 – 1571.
Kume, K., Kume, S., Park, S.K., Hirsh, J., Jackson, F.R., 2005. Dopamine is a
regulator of arousal in the fruit fly. J. Neurosci. 25, 7377 – 7384.
LeBeau, F.E., El Manira, A., Griller, S., 2005. Tuning the network: modulation of
neuronal microcircuits in the spinal cord and hippocampus. Trends Neurosci. 28,
552 – 561.
Lee, C.Y., Yau, S.M., Liau, C.S., Huang, W.J., 2000. Serotonergic regulation of
blood glucose levels in the crayfish, Procamarus clarkii: Site of action and receptor
characterization. J. Exp. Zool. 286, 596 – 605.
Lenke, R.R., Levy, H.R., 1980. Maternal phenylketonuria and
hyperphenylalaninemia: an international survey of the outcome of untreated and
treated pregnancies. N. Engl. J. Med. 303, 1202 – 1208.
Li, H., Cooper, R.L., 2001. Effects of the ecdysoneless mutant on synaptic efficacy
and structure at the neuromuscular junction in Drosophila larvae during normal and
prolonged development. Neurosci. 106, 193 – 200.
177
Li, H., Peng, X., Cooper, R.L., 2002. Development of Drosophila larval
neuromuscular junctions: Maintaining synaptic strength. Neurosci. 115, 505 – 513.
McMahon, L.R., Filip, M., Cunningham, K.A., 2001. Differential regulation of the
mesoaccumbens circuit by serotonin 5-hydroxytryptamine (5-HT)2A and 5-HT2C
receptors. J. Neurosci. 21, 7781 – 7787.
Mentre, F., Escolano, S., 2006. Prediction discrepancies for the evaluation of
nonlinear mixed-effects models. J. Pharmacokinet. Pharmacodyn. 33, 345 – 367.
Monastirioti, M., 1999. Biogenic amine systems in the fruit fly Drosophila
melanogaster. Microsc. Res. Tech. 45, 106 – 121.
Nassel, D.R., 2002. Neuropeptides in the nervous system of Drosophila and other
insects: multiple roles as neuromodulators and neurohormones.
Prog. Neurobiol. 68, 1 – 84.
Neckameyer, W.S., Cooper, R.L., 1998. GABA transporters in Drosophila
melanogaster: developmental expression, behavior, and physiology. Invert.
Neurosci. 3, 279 – 294.
Neckameyer, W.S., 1996. Multiple roles for dopamine in Drosophila development.
Dev. Biol. 176, 209 – 219.
178
Neckameyer, W.S., 1998a. Dopamine and mushroom bodies in Drosophila:
experience-dependent and -independent aspects of sexual behavior. Learn Mem. 5
(1-2), 157 – 165.
Neckameyer, W.S., 1998b. Dopamine modulates female sexual receptivity in
Drosophila melanogaster. J. Neurogenet. 12,101 – 114.
Nichols, C.D., Ronesi, J., Pratt, W., Sanders-Bush, E., 2002. Hallucinogens and
Drosophila: linking serotonin receptor activation to behavior. Neurosci. 115, 979 –
984.
Nichols, R., Kaminski, S., Walling, E., Zornik, E., 1999. Regulating the activity of a
cardioacceleratory peptide. Peptides. 20, 1153 – 1158.
Nuttley, W.M., Atkinson-Leadbeater, K.P., Van Der Kooy, D., 2002. Serotonin
mediates food-odor associative learning in the nematode Caenorhabditis elegans.
Proc. Natl. Acad. Sci. U. S. A. 99, 12449 – 12454.
Osborne, R.H., 1996. Insect neurotransmission: neurotransmitters and their
receptors. Pharmacol. Ther. 69, 117 – 142.
Pauwels, P.J., 2000. Diverse signalling by 5-hydroxytryptamine (5-HT) receptors.
Biochem. Pharmacol. 60, 1743 – 1750.
179
Payne, F., 1911. Drosophila ampelophila bred in the dark for sixty-nine generations.
Biological Bulletin. 21, 297 – 301.
Penzlin, H., 1985. Stomatogastric nervous system. In Kerkut, G. A. (ed),
Comprehensive Insect Physiology Biochemistry and Pharmacology. Oxford:
Pergamon Press. 371 – 406.
Roach, J.L., Wiersma, C.A., 1974. Differentiation and degeneration of crayfish
photoreceptors in darknes. Cell Tissue Res. 153,137 – 144.
Roux, C., Madani, M., Launay, J., Rey, F., Citadelle, D., Mulliez, N., Kolf, M., 1995.
Serotonin defciency in henylketonuria embryopathy. Toxicol. in Vitro, 9, 653 – 662.
Sandeman, D., Beltz, B., Sandeman, R., 1995. Crayfish brain interneurons that
converge with serotonin giant cells in accessory lobe glomeruli. J. Comp. Neurol.
352, 263 – 279.
Scott, E.K., Reuter, J.E, Luo, L., 2003. Dendritic development of Drosophila high
order visual system neurons is independent of sensory experience. BMC Neurosci.
30, 4 – 14.
180
Sewell, D., Burnet, B., Connolly, K., 1975. Genetic analysis of larval feeding
behavior in Drosophlia melanogaster. Gene.t Res. Camb. 24, 163 – 173.
Shuranova, Z.P., Burmistrov, Y.M., Strawn, J.R., Cooper, R.L., 2006. Evidence for
an Autonomic Nervous System in Decapod Crustaceans. Int. J. Zool. Res. 2, 242 –
283.
Simantov, R., 2004. Multiple molecular and neuropharmacological effects of MDMA
(Ecstasy). Life Sci. 74, 803 – 814.
Sodhi, M.S., Sanders-Bush, E., 2004. Serotonin and brain development. Int. Rev.
Neurobiol. 59, 111 – 174.
Sparks, G.M., Brailoiu, E., Brailoiu, C., Dun, N.J., Tabor, J., Cooper, R.L., 2003.
Effects of m-CPP in altering neuronal function: Blocking depolarization in
invertebrate motor & sensory neurons but exciting rat dorsal root neurons. Brain
Res., 969, 14 – 26.
Sprague, J.E., Nichols, D.E., 2005. Neurotoxicity of MDMA (ecstasy): beyond
metabolism. Trends Pharmacol. Sci. 26, 59 – 60.
181
Stewart, B.A., Atwood, H.L., Renger, J.J., Wang, J., Wu, C.F., 1994. Improved
stability of Drosophila larval neuromuscular preparation in hemolymph-like
physiological solutions. J. Comp. Physiol. A. 175, 179 – 191.
Strawn, J.R., Neckameyer, W.S., Cooper, R.L., 2000. The effects of 5-HT on
sensory, central and motor neurons driving abdominal superficial flexor muscles in
the crayfish. Comp. Biochem. Physiol. B. 127, 533 – 550.
Tiernery, A.J., 2001. Structure and function of invertebrate 5-HT receptors: a review.
Comp. Biochem. Physiol. A. Mol. Integr. Physiol. 128, 791 – 804.
Toth, A.L., Kantarovich, S., Meisel, A.F., Robinson, G.E., 2005. Nutritional status
influences socially regulated foraging ontogeny in honey bees. J. Exp. Biol. 208,
4641 – 4649.
Valles, A.M., White, K., 1988. Serotonin-containing neurons in Drosophila
melanogaster: development and distribution. J. Comp. Neurol. 268, 414 – 428.
Vitalis, T., Parnavelas, J.G., 2003. The role of serotonin in early cortical
development. Dev. Neurosci. 25, 245 – 256.
Walther, D.J., Peter, J.U., Bashammakh, S., Hortnagl, H., Voits, M., Fink, H.,
Bader, M., 2003. Synthesis of serotonin by a second tryptophan hydroxylase
isoform. Science. 299, 76 – 76.
182
Weiger, W.A., 1997. Serotonergic modulation of behaviour: a phylogenetic
overview. Biol. Rev. Camb. Philos. Soc. 72, 61 – 95.
Zavarzin, A.A., 1941 Ocherki po evol'utsionnoj gistologii nervnoj sistemy (Essays on
the evolutionary histology of the nervous system). In Zavarzin, A.A. Izbrannye trudy
(Selected Works), Tom III, Izdatel'stvo AN SSSR: Moskva-Leningrad, 1950. [in
Russian]
CHAPTER 4 Barnes, N.M., Sharp, T., 1999. A review of central 5-HT receptors and their function.
Neuropharmacol. 38, 1083 – 1152.
Blenau, W., Baumann, A., 2001. Molecular and pharmacological properties of insect
biogenic amine receptors: Lessons from Drosophila melanogaster and Apis
mellifera. Arch. Insect Biochem. Physiol. 48, 13 – 38.
Bossing, T., Brand, A.H., 2002. Dephrin, a transmembrane ephrin with a unique
structure, prevents interneuronal axons from exiting the Drosophila embryonic CNS.
Development. 129, 4205 – 4218.
Bossing, T., Brand, A.H., 2006. Determination of cell fate along the anteroposterior
axis of the Drosophila ventral midline. Development. 133, 1001 –1012.
183
Brand, A., 1999. GFP as a cell and developmental marker in the Drosophila nervous
system. Methods Cell Biol. 58, 165 – 181.
Brand, A.H., Perrimon, N., 1993. Targeted gene expression as a means of altering
cell fates and generating dominant phenotypes. Development. 118, 401 – 415.
Buznikov, G.A., Peterson, R.E., Nikitina, L.A., Bezuglov, V.V., Lauder, J.M., 2005.
The pre-nervous serotonergic system of developing sea urchin embryos and larvae:
pharmacologic and immunocytochemical evidence. Neurochem Res. 30, 825 – 837.
Campos-Ortega, J.A., Hartenstein, V., (Eds.), 1985. In: The embryonic development
of Drosophila melanogaster. Spring-Verlag, Berlin.
Capela, J.P., Ruscher, K., Lautenschlager, M., Freyer, D., Dirnagl, U., Gaio, A.R.,
Bastos, M.L., Meisel, A., Carvalho, F., 2006. Ecstasy-induced cell death in cortical
neuronal cultures is serotonin 2A-receptor-dependent and potentiated under
hyperthermia. Neurosci. 139, 1069 – 1081.
Cash, S., Chiba, A., Keshishian, H., 1992. Alternate neuromuscular target selection
following the loss of single muscle fibers in Drosophila. J. Neurosci. 12, 2051 –
2064.
184
Choi, D.S., Maroteaux, L., 1996. Immunohistochemical localisation of the serotonin
5-HT2B receptor in mouse gut, cardiovascular system, and brain.
FEBS. Lett. 391, 45 – 51.
Choi, D.S., Ward, S.J., Messaddeq, N., Launay, J.M., Maroteaux, L., 1997. 5-HT2B
receptor-mediated serotonin morphogenetic functions in mouse cranial neural crest
and myocardiac cells. Development. 124, 1745 – 1755 .
Colas, J.F., Launay, J.M., Kellermann, O., Rosay, P., Maroteaux, L., 1994.
Drosophila 5-HT2 serotonin receptor: coexpression with fushi-tarazu during
segmentation, Proc. Natl. Acad. Sci. U. S. A. 92, 5441 – 5445.
Colas, J.F., Launay, J.M., Maroteaux, L., 1999. Maternal and zygotic control of
serotonin biosynthesis are both necessary for Drosophila germband extension.
Mech. Dev. 87, 67 – 76.
Colas, J.F., Launay, J.M., Vonesch, J.L., Hickel, P., Maroteaux, L., 1999. Serotonin
synchronises convergent extension of ectoderm with morphogenetic gastrulation
movements in Drosophila. Mech Dev. 87, 77 – 91.
Cooper, R.L., Stewart, B.A., Wojtowicz, J.M., Wang, S., Atwood, H.L., 1995. Quantal
measurement and analysis methods compared for crayfish and Drosophila
neuromuscular junctions, and rat hippocampus, J. Neurosci. Methods. 61, 67 – 78.
185
Cote, F., Thevenot, E., Fligny, C., Fromes, Y., Darmon, M., Ripoche, M.A., Bayard,
E., Hanoun, N., Saurini, F., Lechat, P., Dandolo, L., Hamon, M., Mallet, J., Vodjdani,
G., 2003. Disruption of the nonneuronal tph1 gene demonstrates the importance of
peripheral serotonin in cardiac function.
Proc. Natl. Acad. Sci. U. S. A. 100, 13525 – 13530.
Dasari, S., Cooper, R.L., 2004. Modulation of sensory to motor circuits by serotonin,
octopamine, and dopamine in semi-intact Drosophila larva. Neurosci. Res. 48, 221 –
227.
Dasari, S., Cooper, R.L., 2006. Direct influence of serotonin on the larval heart of
Drosophila melanogaster. J. Comp. Physiol. B. 176, 349 – 357.
Dasari, S., Turner, A.C., Cullman-Clark, B., White, J., Cooper, R. L., 2006. Effects of
the serotonergic system on physiology, development, learning and behavior of
Drosophila melanogaster. Society for neurosci. abst. (Georgia World Congress
Center) 427, 14 / C78.
Frishman, W.H., Grewall, P., 2000. Serotonin and the heart.
Ann. Med. 32, 195 – 209.
186
Fuller, R.W., 1991. Role of serotonin in therapy of depression and related disorders.
J. Clin. Psychiatry. 52, Suppl, 52 – 57.
Fussnecker, B.L., Smith, B.H., Mustard, J.A., 2006. Octopamine and tyramine
influence the behavioral profile of locomotor activity in the honey bee (Apis
mellifera). J. Insect Physiol. 52, 1083 – 1092.
Hoyer, D., Hannon, J.P., Martin, G.R., 2002. Molecular, pharmacological and
functional diversity of 5-HT receptors. Pharmacol. Biochem. Behav. 71, 533 – 554.
Iyengar, B.G., Chou, C.J., Sharma, A., Atwood, H.L., 2006. Modular neuropile
organization in the Drosophila larval brain facilitates identification and mapping of
central neurons. J. Comp. Neurol. 499, 583 – 602.
Johnson, E., Ringo, J., Dowse, H., 1997. Modulation of Drosophila heartbeat by
neurotransmitters. J. Comp. Physiol. B. 167, 89 – 97.
Johnstone, A.F.M., Cooper, R.L., 2006. Direct innervation of the Drosophila
melanogaster larval heart. Brain Res. 1083, 159 – 163.
Kendler, K.S., Greenspan, R.J., 2006. The nature of genetic influences on behavior:
lessons from "simpler" organisms. Am. J. Psychiatry. 163, 1683 – 1694.
187
Lauder, J.M., Wilkie, M.B., Wu. C., Singh, S., 2000. Expression of 5-HT (2A), 5-
HT(2B) and 5-HT(2C) receptors in the mouse embryo. Int. J. Dev. Neurosci. 18, 653
– 662.
Lee, T., Luo, L., 2001. Mosaic analysis with a repressible cell marker (MARCM) for
Drosophila neural development. Trends Neurosci. 24, 251 – 254.
Leonard, B.E., 1994. Serotonin receptors--where are they going?
Int Clin Psychopharmacol. 9 Suppl 1, 7 – 17.
Li, H., Cooper, R.L., 2001. Effects of the ecdysoneless mutant on synaptic efficacy
and structure at the neuromuscular junction in Drosophila larvae during normal and
prolonged development. Neurosci. 106, 193 – 200.
Li, H., Harrison, D., Jones, G., Jones, D., Cooper, R.L., 2001. Alterations in
development, behavior, and physiology in Drosophila larva that have reduced
ecdysone production, J. Neurophysiol. 85, 98 – 104.
Lucki, I., 1998. The spectrum of behaviors influenced by serotonin. Biol. Psychiatry.
44, 151 – 162.
MacLean, M.R., Clayton, R.A., Templeton, A.G., Morecroft, I., 1996. Evidence for 5-
HT1-like receptor-mediated vasoconstriction in human pulmonary artery.
Br. J. Pharmacol. 119, 277 – 282.
188
Monastirioti, M., 1999. Biogenic amine systems in the fruit fly Drosophila
melanogaster. Microscopy Res. Tech. 45, 106 – 121.
Nassif, C., Noveen, A., Hartenstein, V., 2003. Early development of the Drosophila
brain: III. The pattern of neuropile founder tracts during the larval period. J. Comp.
Neurol. 20, 455, 417 – 434.
Nebigil, C.G., Choi, D.S., Dierich, A., Hickel, P., Le Meur, M., Messaddeq, N.,
Launay, J.M., Maroteaux, L., 2000. Serotonin 2B receptor is required for heart
development. Proc. Natl. Acad. Sci. U. S. A. 97, 9508 – 9513.
Nebigil, C.G., Hickel, P., Messaddeq, N., Vonesch, J.L., Douchet, M.P., Monassier,
L., Gyorgy, K., Matz, R., Andriantsitohaina, R., Manivet, P., Launay, J.M.,
Maroteaux, L., 2001. Ablation of serotonin 5-HT(2B) receptors in mice leads to
abnormal cardiac structure and function.
Circulation. 103, 2973 – 2979.
Neckameyer, W.S., 1996. Multiple roles for dopamine in Drosophila development.
Dev. Biol. 176, 209 – 219.
189
Neckameyer, W.S., Cooper, R.L., 1998. GABA transporters in Drosophila
melanogaster: molecular cloning, behavior, and physiology, Invert. Neurosci. 3, 279
– 294.
Osborne, R. H., 1996. Insect Neurotransmission: Neurotransmitters and Their
Receptors, Pharmacol. Ther. 69, 117 – 142.
Pauly, J.R., Turner, A.C., Cooper, R.L., 2006. Influence of the dopamine on
physiology, development, and behavior of Drosophila melanogaster. Society for
Neurosci. Abst. 427.13.
Rapport, M.M., Green, A.A., Page, I.H., 1948. Partial purification of the
vasoconstrictor in beef serum. J. Biol. Chem. 174, 735 – 738.
Reynolds, G.P., Templeman, L.A., Zhang, Z.J., 2005. The role of 5-HT2C receptor
polymorphisms in the pharmacogenetics of antipsychotic drug treatment. Prog.
Neuropsychopharmacol Biol. Psychiatry. 29, 1021 – 1028.
Roth, B.L., Willins, D.L., Kristiansen, K., Kroeze, W.K., 1988. 5-Hydroxytryptamine2-
family receptors (5-hydroxytryptamine2A, 5-hydroxytryptamine2B, 5-
hydroxytryptamine2C): where structure meets function.
Pharmacol. Ther. 79, 231 – 257.
190
Saudou, F., Boschert, U., Amlaiky, N., Plassat, J.L., Hen, R. 1992. A family of
Drosophila serotonin receptors with distinct intracellular signaling properties and
expression patterns. J. EMBO. 11, 7 – 17.
Sewell, D.F., Hunt, D.M., Burnet, B.,1975. Biogenic amines in Drosophila
melanogaster selected for differences in larval feeding behavior. Behav. Biol. 15,
213 – 217.
Shupliakov, O., Bloom, O., Gustafsson, J.S., Kjaerulff, O., Low, P., Tomilin, N.,
Pieribone, V.A., Greengard, P., Brodin, L., 2002. Impaired recycling of synaptic
vesicles after acute perturbation of the presynaptic actin cytoskeleton. Proc. Natl.
Acad. Sci. U. S. A. 99, 14476 – 14481.
Sokolowski, M.B., 1980. Foraging strategies of Drosophila melanogaster: a
chromosomal analysis. Behav. Genet. 10, 291 – 302.
Stewart, B.A., Atwood, H.L., Renger, J.J., Wang, J., Wu, C.F., 1994. Improved
stability of Drosophila larval neuromuscular preparation in haemolymph-like
physiological solutions, J. Comp. Physiol. A. 175 ,179 – 191.
Tierney, A.J., 2001. Structure and function of invertebrate 5-HT receptors: a review.
Comp. Biochem. Physiol. A. 128, 791 – 804.
191
Valles, A.M., White, K., 1988. Serotonin-containing neurons in Drosophila
melanogaster: development and distribution. J. Comp. Neurol. 268, 414 – 428.
Whitaker-Azmitia, P.M., 2001. Serotonin and brain development: role in human
developmental diseases. Brain Res. Bull. 56, 479 – 485.
Witz, P., 1990. Cloning and chacterization of a Drosophila serotonin receptor that
activates adenylate cyclase. Neurobiol. 87, 8940 – 8944.
Younossi-Hartenstein, A., Salvaterra, P.M., Hartenstein, V., 2003. Early
development of the Drosophila brain: IV. Larval neuropile compartments defined by
glial septa. J. Comp. Neurol. 455, 435 – 450.
Yuan, Q., Joiner, W.J., Sehgal, A., 2006. A sleep-promoting role for the Drosophila
serotonin receptor 1A. Curr. Biol. 16, 1051 – 1062.
Zornik, E., Paisley, K., Nichols, R., 1999. Neural transmitters and a peptide modulate
Drosophila heart rate. Peptides. 20, 45 – 51.
CHAPTER 5 Ashton, K., Wagoner, A.P., Carrillo, R., Gibson, G., 2001. Quantitative trait loci for
the monoamine-related traits heart rate and headless behavior in Drosophila
melanogaster. Genetics 157, 283 – 294.
192
Badre, N.H., Martin, M.E., Cooper, R.L., 2005. The physiological and behavioral
effects of carbon dioxide on Drosophila melanogaster larvae. Comp. Biochem.
Physiol. A. Mol. Integr. Physiol. 140, 363 – 376.
Ball, R., Xing, B., Bonner, P., Shearer, J., Cooper, R.L., 2003. Long-term
maintenance of neuromuscular junction activity in cultured Drosophila larvae. Comp.
Biochem. Physiol. Mol. Integr. Physiol. 134, 247 – 255.
Campos-Ortega, J.A., Hartenstein, V., (eds) 1985. In: The embryonic development
of Drosophila melanogaster. Spring-Verlag, Berlin. 2nd edition, pp. 1 – 227.
Colas, J.F., Launay, J.M., Kellermann, O., Rosay, P., Maroteaux, L., 1995.
Drosophila 5-HT2 serotonin receptor: coexpression with fushi-tarazu during
segmentation. Proc. Natl. Acad. Sci. U. S. A. 92, 5441 – 5445.
Cooper, R.L., Neckameyer, W.S., 1999. Dopaminergic modulation of motor neuron
activity and neuromuscular function in Drosophila melanogaster. Comp. Biochem.
Physiol. B. Biochem. Mol. Biol. 122, 199 – 210.
Dasari, S., Cooper, R.L., 2004. Modulation of sensory to motor circuits by serotonin,
octopamine, and dopamine in semi-intact Drosophila larva. Neurosci. Res. 48, 221 –
227.
193
Dasari, S., Cooper, R.L., 2005. Monitoring heart rate in Drosophila larvae by various
approaches. Drosophila information Service. 87, 91 – 96.
Dasari, S., Nichols, R., Cooper, R.L., 2004. Effects of 5-HT and MDMA on heart rate
of 3rd instar Drosophila melanogaster. Program No. 466.13. Washington, DC:
Society for Neuroscience, 2004.
De La Torre, R., Farre, M., 2004. Neurotoxicity of MDMA (ecstasy): the limitations of
scaling from animals to humans. Trends Pharmacol. Sci. 25, 505 –
508.
Djokaj, S., Cooper, R.L., Rathmayer, W., 2001. Effects of octopamine, serotonin,
and cocktails of the two modulators on synaptic transmission at crustacean
neuromuscular junctions. J. Comp. Physiol. A. 187, 145 – 154.
Dowse, H., Ringo, J., Power, J., Johnson, E., Kinney, K., White, L., 1995. A
congenital heart defect in Drosophila caused by an action-potential mutation. J.
Neurogenet. 10, 153 – 168.
Dulcis, D., Levine, R.B., 2003. Innervation of the heart of the adult fruit fly,
Drosophila melanogaster. J. Comp. Neurol. 27, 560 – 578.
194
Dulcis, D., Levine, R.B., 2005. Glutamatergic innervation of the heart initiates
retrograde contractions in adult Drosophila melanogaster. J. Neurosci. 12, 271 –
280.
Dulcis, D., Levine, R.B., Ewer, J., 2005. Role of the neuropeptide CCAP in
Drosophila cardiac function. J. Neurobiol. 5, 259 – 274.
Fitzgerald, L.W., Burn, T.C., Brown, B.S., Patterson, J.P., Corjay, M.H., Valentine,
P.A., Sun, J.H., 2000. Possible role of valvular serotonin 5-HT(2B) receptors in the
cardiopathy associated with fenfluramine. Mol. Pharmacol. 57, 75 – 81.
Fowler, D.J., Goodnight, C.J., Labrie, M.M., 1972. Circadian rhythms of 5-
hydrosytryptamine (serotonin) production in larvae, pupae, and adults of Drosophila
melanogaster (Diptera:Drosophilidae). Ann. Entomol. Soc. Am. 65, 138 – 141.
Green, A.R., Mechan, A.O., Elliott, J.M., O'Shea, E., Colado, M.I., 2003. The
pharmacology and clinical pharmacology of 3,4 methylenedioxy-methamphetamine
(MDMA, "ecstasy"). Pharmacol. Rev. 55, 463 – 508.
Gregg, T.G., McCrate, A., Reveal, G., Hall, S., Rypstra, A.L., 1990. Insectivory and
social digestion in Drosophila. Biochem. Genet. 28, 197 – 207.
195
Gu, G.G., Singh, S., 1995. Pharmacological analysis of heartbeat in Drosophila.
J. Neurobiol. 28, 269 – 280.
He, B., Adler, P.N., 2001. Cellular mechanisms in the development of the Drosophila
arista. Mech. Dev. 104, 69 – 78.
Hirsh, J., 2001. Time flies like an arrow. Fruit flies like crack? Pharmacogenomics.
J. 1, 97 – 100.
Hirashima, A., Sukhanova, M.Jh., Rauschenbach, I.Yu., 2000. Biogenic amines in
Drosophila virilis under stress conditions. Biosci. Biotechnol. Biochem. 64, 2625 –
2630.
Jian, B., Xu, J., Savani, R.C., Narula, N., Liang, B., Levy, R.J., 2002. Serotonin
mechanisms in heart valve disease. I. Serotonin-induced up-regulation of TGF-ß1
via G-protein signal transduction in aortic valve interstitial cells. Am. J. Pathol. 161,
2111 – 2121.
Johnstone, A.F., Cooper, R.L., 2006. Direct innervation of the Drosophila
melanogaster larval aorta. Brain Res. 1083, 159 – 163.
Johnson, E., Ringo, J., Dowse, H., 1997. Modulation of Drosophila heartbeat by
neurotransmitters. J. Comp. Physiol. B. 167, 89 – 97.
196
Johnson, E., Ringo, J., Dowse, H., 2000. Native and heterologous neuropeptides are
cardioactive in Drosophila melanogaster. J. Insect. Physiol. 1, 1229 – 1236.
Johnson, E., Sherry, T., Ringo, J., Dowse, H., 2002. Modulation of the cardiac
pacemaker of Drosophila: cellular mechanisms. J. Comp. Physiol. B. 172, 227 –236.
Lee, C.J., Kong, H., Manzini, M.C., Albuquerque, C., Chao, M.V., MacDermott, A.B.,
2001. Kainate receptors expressed by a subpopulation of developing nociceptors
rapidly switch from high to low Ca2+ permeability. J. Neurosci. 21, 4572 – 4581.
Li, H., Cooper, R.L., 2001. Effects of the ecdysoneless mutant on synaptic efficacy
and structure at the neuromuscular junction in Drosophila larvae during normal and
prolonged development. Neurosci. 106, 193 – 200.
Li, H., Listerman, L.R., Doshi, D., Cooper, R.L., 2000. Heart rate measures in blind
cave crayfish during environmental disturbances and social interactions. Comp.
Biochem. Physiol. A. Mol. Integr. Physiol. 127, 55 – 70.
Listerman, L.R., Deskins, J., Bradacs, H., Cooper, R.L., 2000. Heart rate within male
crayfish: social interactions and effects of 5-HT. Comp. Biochem. Physiol. A. Mol.
Integr. Physiol. 125, 251 – 263.
197
Lisi, T.L., Westlund, K.N., Sluka, K.A., 2003. Comparison of microdialysis and push–
pull perfusion for retrieval of serotonin and norepinephrine in the spinal cord dorsal
horn. J. Neurosci. Methods. 126, 187 – 194.
McCann, U.D., Slate, S.O., Ricaurte, G.A., 1996. Adverse reactions with 3,4-
methylenedioxymethamphetamine (MDMA; 'ecstasy'). Drug Saf. 15, 107 – 115.
Mesce, K.A., 2002. Metamodulation of the biogenic amines: second-order
modulation by steroid hormones and amine cocktails. Brain. Behav. Evol. 60, 339 –
349.
Miller, T. A., 1985. In: Kerkut GA, Gilbert LI (Eds.) Comprehensive Insect
Physiology, Biochemistry and Pharmacology. Pergamon Press, Oxford. pp, 289 –
353.
Miller, T.A., 1997. Control of circulation in insects. Gen. Pharmacol. 29, 23 – 38.
Molina, M.R., Cripps, R.M., 2001. Ostia, the inflow tracts of the Drosophila heart,
develop from a genetically distinct subset of cardial cells. Mech. Dev. 109, 51 –
59.
Monastirioti, M., 1999. Biogenic amine systems in the fruit fly Drosophila
melanogaster. Microsc. Res. Tech. 45, 106 – 121.
198
Neckameyer, W., O'Donnell, J., Huang, Z., Stark, W., 2001. Dopamine and sensory
tissue development in Drosophila melanogaster. J. Neurobiol. 47, 280 –
294.
Nichols, R., Kaminski, S., Walling, E., Zornik, E., 1999. Regulating the activity of a
cardioacceleratory peptide. Peptides. 20, 1153 – 1158.
Papaefthimiou, C., Theophilidis, G., 2001. An in vitro method for recording the
electrical activity of the isolated heart of the adult Drosophila melanogaster.
In Vitro Cell Dev. Biol. Anim. 37, 445 – 449.
Paupard, M.C., O'Connell, M.A., Gerber, A.P., Zukin, R.S., 2000. Patterns of
developmental expression of the RNA editing enzyme rADAR2. Neurosci. 95, 869 –
879.
Peroutka, S.J., Newman, H., Harris, H., 1988. Subjective effects of 3,4-
methylenedioxymethamphetamine in recreational users. Neuropsychopharmacol. 1,
273 – 277.
Ponzielli, R., Astier, M., Chartier, A., Gallet, A., Therond, P., Semeriva, M., 2002.
Heart tube patterning in Drosophila requires integration of axial and segmental
information provided by the Bithorax Complex genes and hedgehog signaling.
Development. 129, 4509 – 4521.
199
Rizki, T., 1978. The circulatory system and associated tissues. In: Ashburner M,
Wright T (eds) The genetics and biology of Drosophila. Academic Press, London,
pp 397 – 452.
Rothenfluh, A., Heberlein, U., 2002. Drugs, flies, and videotape: the effects of
ethanol and cocaine on Drosophila locomotion. Curr. Opin. Neurobiol. 12, 639 –
645.
Roy, A., Brand, N.J., Yacoub, M.H., 2000. Expression of 5-hydroxytryptamine
receptor subtype messenger RNA in interstitial cells from human heart valves. J.
Heart. Valve. Dis. 9, 256 – 260.
Saudou, F., Boschert, U., Amlaiky, N., Plassat, J.L., Hen, R., 1992. A family of
Drosophila serotonin receptors with distinct intracellular signalling properties and
expression patterns. J. EMBO. 11, 7 – 17.
Schapker, H., Breithaupt, T., Shuranova, Z., Burmistrov, Y., Cooper, R.L., 2002.
Heart and ventilatory measures in crayfish during environmental disturbances and
social interactions. Comp. Biochem. Physiol. A. Mol. Integr. Physiol. 131, 397 – 407.
Sláma, K., Farkaš, R., 2005. Heartbeat patterns during the postembryonic
development of Drosophila melanogaster. J. Insect. Physiol. 51, 489 – 503.
200
Slominski, A., Pisarchik, A., Zbytek, B., Tobin, D.J., Kauser, S., Wortsman, J., 2003.
Functional activity of serotoninergic and melatoninergic systems expressed in the
skin. J. Cell. Physiol. 196, 144 – 153.
Southard, R.C., Haggard, J., Crider, M.E., Whiteheart, S.W., Cooper, R.L., 2000.
Influence of serotonin on the kinetics of vesicular release. Brain. Res. 871, 16 –28.
Sparks, G.M., Dasari, S., Cooper, R.L., 2004. Presynaptic actions of MDMA
(Ecstasy) at glutamatergic synapses. Neurosci. Res. 48, 431 – 438.
Sparks, G.M., Brailoiu, E., Brailoiu, C., Dun, N.J., Tabor, J., Cooper, R.L., 2003.
Effects of m-CPP in altering neuronal function: Blocking depolarization in
invertebrate motor & sensory neurons but exciting rat dorsal root neurons. Brain.
Res. 969, 14 – 26.
Stewart, B.A., Atwood, H.L., Renger, J.J., Wang, J., Wu, C.F., 1994. Improved
stability of Drosophila larval neuromuscular preparation in haemolymph-like
physiological solutions. J. Comp. Physiol. A. 175, 179 – 191.
Strawn, J.R., Ekhator, N.N., Geracioti, T.D. Jr, 2001. In-use stability of monoamine
metabolites in human cerebrospinal fluid. J. Chromatogr. B. Biomed. Sci. Appl. 760,
301 – 306.
Visiers, I., Hassan, S.A., Weinstein, H., 2001. Differences in conformational
properties of the second intracellular loop (IL2) in 5-HT(2C) receptors modified by
201
RNA editing can account for G protein coupling efficiency.
Protein. Eng. 14, 409 – 414.
Wessells, R.J., Bodmer, R., 2004. Screening assays for heart function mutants in
Drosophila. Biotechniques. 37, 58 – 60, 62, 64.
Wessells, R.J., Fitzgerald, E., Cypser, J.R., Tatar, M., Bodmer, R., 2004. Insulin
regulation of heart function in aging fruit flies. Nat. Genet. 36, 1275 – 1281.
White, L.A., Ringo, J.M., Dowse, H.B., 1992. Effects of deuterium oxide and
temperature on heart rate in Drosophila melanogaster. J. Comp. Physiol. B. 162,
278 – 283.
Witz, P., Amlaiky, N., Plassat, J.L., Maroteaux, L., Borrelli, E., Hen, R., 1990.
Cloning and characterization of a Drosophila serotonin receptor that activates
adenylate cyclase. Proc. Natl. Acad. Sci. U. S. A. 87, 8940 – 8944.
Wolf, F.W., Heberlein, U., 2003. Invertebrate models of drug abuse. J. Neurobiol. 54,
161 – 178.
Zornik, E., Paisley, K., Nichols, R., 1999. Neural transmitters and a peptide modulate
Drosophila heart rate. Peptides. 20, 45 – 51.
202
CHAPTER 6
Alshuaib, W.B., Mathew, M.V., Hasan, M.Y., Fahim, M.A., 2003. Serotonin reduces
potassium current in rutabaga and wild-type Drosophila neurons. Int. J. Neurosci.
113, 1413 – 1425.
Baier, A., Wittek, B., Brembs, B., 2002. Drosophila as a new model organism for the
neurobiology of aggression? J. Exp. Biol. 205, 1233 – 1240.
Banerjee, S., Lee, J., Venkatesh, K., Wu, C.F., Hasan, G., 2004. Loss of flight and
associated neuronal rhythmicity in inositol 1,4,5-trisphosphate receptor mutants of
Drosophila. J. Neurosci. 24, 7869 – 7878.
Borbely, A.A., Huston, J.P., Waser, P.G., 1973. Physiological and behavioral effects
of parachlorophenylalanine in the rat. Psychopharmacol. 31, 131 – 142 .
Broening, H.W., Morford, L.L., Inman-Wood, S.L., Fukumura, M., Vorhees, C.V.,
2001. 3,4-methylenedioxymethamphetamine (ecstasy)-induced learning and
memory impairments depend on the age of exposure during early development.
J. Neurosci. 21, 3228 – 3235.
Budnik, V., White, K., 1987a. Genetic dissection of dopamine and serotonin
synthesis in the nervous system of Drosophila melanoguster. J. Neurogenet. 4, 309
– 314.
203
Budnik, V., White, K., 1987b. A serotonergic system in Drosophila melanogasterr.
Soc. Neurosci. Abstr. 13, 234.
Butkevich, I.P., Khozhai, L.I., Mikhailenko, V.A., Otellin, V.A., Decreased serotonin
level during pregnancy alters morphological and functional characteristics of tonic
nociceptive system in juvenile offspring of the rat. Reprod. Biol. Endocrinol. 1, 96.
Callaway, C.W., Wing, L.L., Geyer, M.A., 1990. Serotonin release contributes to the
locomotor stimulant effects of 3,4-methylenedioxymethamphetamine in rats. J.
Pharmacol. Exp. Ther. 254, 456 – 464.
Cnaani, J., Robinson, G.E., Hefetz, A., 2000. The critical period for caste
determination in Bombus terrestris and its juvenile hormone correlates. J. Comp.
Physiol. A. 186, 1089 – 1094.
Colas, J.F., Launay, J.M., Maroteaux, L., 1999. Maternal and zygotic control of
serotonin biosynthesis are both necessary for Drosophila germband extension.
Mech. Dev. 87, 67 – 76.
Coleman, C.M., Neckameyer, W.S., 2005. Serotonin synthesis by two distinct
enzymes in Drosophila melanogaster. Arch. Insect Biochem. Physiol. 59, 12 – 31.
Cote, F., Thevenot, E., Fligny, C., Fromes, Y., Darmon, M., Ripoche, M.A., Bayard,
E., Hanoun, N., Saurini, F., Lechat, P., Dandolo, L., Hamon, M., Mallet, J., Vodjdani,
204
G., 2003. Disruption of the nonneuronal tph1 gene demonstrates the importance of
peripheral serotonin in cardiac function. Proc. Natl. Acad. Sci. U. S. A. 100, 13525
– 13530.
Crowell, M.D., 2001. The role of serotonin in the pathophysiology of irritable bowel
syndrome. Am. J. Manag. Care. 7, S 252 – 260. Review.
Daw, N.W., Fox, K., Sato, H., Czepita, D., 1992. Critical period for monocular
deprivation in the cat visual cortex. J Neurophysiol. 67, 197 – 202.
Demchyshyn, L.L., Pristupa, Z.B., Sugamori, K.S., Barker, E.L., Blakely, R.D.,
Wolfgang, W.J., Forte, M.A., Niznik, H.B., 1994. Cloning, expression, and
localization of a chloride-facilitated, cocaine-sensitive serotonin transporter from
Drosophila melanogaster. Proc. Natl. Acad. Sci. U. S. A. 91, 5158 – 5162.
Dringenberg, H.C., Hargreaves, E.L., Baker, G.B., Cooley, R.K., Vanderwolf, C.H.,
1995. p-chlorophenylalanine-induced serotonin depletion: reduction in exploratory
locomotion but no obvious sensory-motor deficits. Behav. Brain Res. 68, 229 – 237.
Dulcis, D., Levine, R.B., 2003. Innervation of the heart of the adult fruit fly,
Drosophila melanogaster. J. Comp. Neurol. 465, 560 – 578.
205
Edagawa, Y., Saito, H., Abe, K., 2001. Endogenous serotonin contributes to a
developmental decrease in long-term potentiation in the rat visual cortex. J.
Neurosci. 21, 1532 - 1537.
Fibiger, H.C., Campbell, B.A., 1971. The effect of para-chlorophenylalanine on
spontaneous locomotor activity in the rat. Neuropharmacol. 10, 25 – 32.
Fickbohm, D.J., Katz, P.S., 2000. Paradoxical actions of the serotonin precursor 5-
hydroxytryptophan on the activity of identified serotonergic neurons in a simple
motor circuit. J Neurosci. 20, 1622 – 1634.
Freedman, R.R., Johanson, C.E., Tancer, M.E., 2005. Thermoregulatory effects of
3,4-methylenedioxymethamphetamine (MDMA) in humans. Psychopharmacol.
(Berl). 183, 248 – 256.
Fox, L.E., Soll, D.R., Wu, C.F., 2006. Coordination and modulation of locomotion
pattern generators in Drosophila larvae: effects of altered biogenic amine levels by
the tyramine beta hydroxlyase mutation. J. Neurosci. 26, 1486 – 1498 .
Gorczyca, M.G., Budnik, V., White, K., Wu, C.F., 1991. Dual muscarinic and nicotinic
action on a motor program in Drosophila. J. Neurobiol. 22, 391 – 404.
206
Green, A.R., Mechan, A.O., Elliott, J.M., O'Shea, E., Colado, M.I., 2003. The
pharmacology and clinical pharmacology of 3,4 methylenedioxy-methamphetamine
(MDMA, "ecstasy"). Pharmacol. Rev., 55, 463 – 508.
Greenspan, R.J., Finn, J.A.Jr., Hall, J.C., 1980. Acetylcholinesterase mutants in
Drosophila and their effects on the structure and function of the central nervous
system. J. Comp. Neurol. 189, 741 – 774.
Gu, S. H., Chow, Y. S., Lin, F. J., Wu, J. L., Ho, R. J., 1996. A deficiency in
prothoracicotropic hormone transduction pathway during the early last larval instar of
Bombyx mori. Mol. Cell. Endocrinol. 120, 99 – 105.
Harris, P., Fritts, H.W., Cournand, A., 1960. Some circulatory effects of 5-
hydroxytryptamine in man. Circulation. 21, 1134 – 1139.
Harris-Warrick, R.M., Cohen, A.H., 1985. Serotonin modulates the central pattern
generator for locomotion in the isolated lamprey spinal cord. J. Exp. Biol. 116, 27 –
46.
Hellendall, R.P., Schambra, U.B., Liu, J.P., Lauder, J.M., 1993. Prenatal expression
of 5-HT1C and 5-HT2 receptors in the rat central nervous system.
Exp. Neurol. 120, 186 – 201.
207
Hollander, W., Michelson, A.L., Wilkins, R.W., 1957. Serotonin and antiserotonins: I.
their circulatory, respiratory and renal effects in man. Circulation. 16, 246 – 255.
Hubel, D.H., Wiesel, T.N., 1970. The period of susceptibility to the physiological
effects of unilateral eye closure in kittens. J. Physiol. 206, 419 – 436.
Jacobs, B.L., Fornal, C.A., 1993. 5-HT and motor control: a hypothesis. Trends
Neurosci. 16, 346 – 352.
Johnstone, A.F.M., Cooper, R.L., 2006. Direct innervation of the Drosophila
melanogaster larval heart. Brain res. 1083, 159 – 163.
Kamyshev, N.G., Smirnova, G.P., Savvateeva, E.V., Medvedeva, A.V.,
Ponomarenko, V.V., 1983. The influence of serotonin and p-chlorophenylalanine on
locomotor activity of Drosophila melanogaster. Pharmacol. Biochem. Behav., 18,
677 – 681.
Katz, P.S., Getting, P.A., Frost, W.N., 1994. Dynamic neuromodulation of synaptic
strength intrinsic to a central pattern generator circuit. Nature. 367, 729 – 731.
Katz, P.S., Harris-Warrick, R.M., 1999. The evolution of neuronal circuits underlying
species-specific behavior. Curr. Opin. Neurobiol. 9, 628 – 633.
208
Kaumann, A.J., Levy, F.O., 2006. 5-hydroxytryptamine receptors in the human
cardiovascular system. Pharmacol. Ther. 111, 674 – 706.
Kehne, J.H., Ketteler, H.J., McCloskey, T.C., Sullivan, C.K., Dudley, M.W., Schmidt,
C.J., 1996. Effects of the selective 5-HT2A receptor antagonist MDL 100,907 on
MDMA-induced locomotor stimulation in rats. Neuropsychopharmacol. 15, 116 –
124.
Koe, B.K., Weissman, A., 1966. p-Chlorophenylalanine: a specific depletor of brain
serotonin. J. Pharmacol. Exp. Ther. 154, 499 – 516.
Kojic, L., Gu, Q., Douglas, R.M., Cynader, M.S., 2001. Laminar distribution of
cholinergic- and serotonergic-dependent plasticity within kitten visual cortex. Brain.
Res. Dev. Brain. Res. 126, 157 – 162.
Kristan, W.B.Jr, Nusbaum, M.P., 1982 – 1983. The dual role of serotonin in leech
swimming. J. Physiol. (Paris). 78, 743 – 747.
Lauder, J.M., Krebs, H., 1978. Serotonin as a differentiation signal in early
neurogenesis. Dev. Neurosci. 1,15 – 30.
209
Li, H., Cooper, R.L., 2001. Maintaining synaptic efficacy at the neuromuscular
junction in Drosophila larva during normal development and prolonged life with the
ecdysoneless mutant. Neurosci. 106, 193 – 200
Listerman, L., Deskins, J., Bradacs, H., Cooper, R.L., 2000. Measures of heart rate
during social interactions in crayfish and effects of 5-HT. Comp. Biochem. Physiol.
A.125, 251 – 264.
Marder, E., Thirumalai, V., 2002, Cellular, synaptic and network effects of
neuromodulation. Neural Networks. 15, 479 – 493.
Marsden, C.A., Curzon, G., 1976. Studies on the behavioural effects of tryptophan
and p-chlorophenylalanine. Neuropharmacol. 15, 165 – 171.
Mas, M., Farre, M., de, la, Torre, R., Roset, P.N., Ortuno, J., Segura, J., Cami, J.,
1999. Cardiovascular and neuroendocrine effects and pharmacokinetics of 3, 4-
methylenedioxymethamphetamine in humans. J. Pharmacol. Exp. Ther. 290, 136 –
145.
Miller, M.R., McClure, D., Shiman, R., 1975. p-Chlorphenylalanine effect on
phenylalanine hydroxylase in hepatoma cells in culture. J. Biol. Chem. 250, 1132 –
1140.
210
Miller, M.R., McClure, D., Shiman, R., 1976. Mechanism of inactivation of
phenylalanine hydroxylase by p-chlorophenylalanine in hepatome cells in culture.
Two possible models. J. Biol. Chem. 251, 3677 – 3684.
Myoga, H., Nonaka, S., Matsuyama, K., Mori, S., 1995. Postnatal development of
locomotor movements in normal and para-chlorophenylalanine-treated newborn rats.
Neurosci. Res. 21, 211 – 221.
Neckameyer, W.S., White, K., 1993. Drosophila tyrosine hydroxylase is encoded by
the pale locus. J. Neurogenet. 8, 189 – 199.
O'Cain, P.A., Hletko, S.B., Ogden, B.A., Varner, K.J., 2000. Cardiovascular and
sympathetic responses and reflex changes elicited by MDMA. Physiol. Behav. 70,
141 – 148.
Palenicek, T., Votava, M., Bubenikova, V., Horacek, J., 2005. Increased sensitivity to
the acute effects of MDMA ("ecstasy") in female rats. Physiol. Behav. 86, 546 – 553.
Pirch, J.H., 1969. Stimulation of locomotor activity by p-chlorophenylalanine and a
low dose of reserpine. Arch. Int. Pharrnacodyn. 181, 434 – 440.
211
Rempel, N.L., Callaway, C.W., Geyer, M.A., 1993. Serotonin1B receptor activation
mimics behavioral effects of presynaptic serotonin release.
Neuropsychopharmacol. 8, 201 – 211.
Rodriguez, Moncalvo, V.G., Campos, A.R., 2005. Genetic dissection of trophic
interactions in the larval optic neuropil of Drosophila melanogaster. Dev. Biol. 286,
549 – 558.
Sanchez, V., Camarero, J., Esteban, B., Peter, M.J., Green, A.R., Colado, M.I.,
2001. The mechanisms involved in the long-lasting neuroprotective effect of
fluoxetine against MDMA ('ecstasy')-induced degeneration of 5-HT nerve endings in
rat brain. Br. J. Pharmacol. 134, 46 – 57.
Setola, V., Dukat, M., Glennon, R.A., Roth, B.L., 2005. Molecular determinants for
the interaction of the valvulopathic anorexigen norfenfluramine with the 5-HT2B
receptor. Mol. Pharmacol. 68, 20 – 33.
Song, Q., Gilbert L. I., 1996. Protein phosphatase activity is required for
prothoracicotropic hormone-stimulated ecdysteroidogenesis in the prothoracic
glands of the tobacco hornworm, Manduca sexta. Arch. Insect Biochem. Physiol. 31,
465 – 480
Stevenson, P. A., Hofmann, H. A., Schoch, K., Schildberger, K., 2000. The fight and
flight responses of crickets depleted of biogenic amines. J. Neurobiol. 43, 107 – 120.
212
Suster, M.L., Bate, M., 2002. Embryonic assembly of a central pattern generator
without sensory input. Nature. 416, 174 – 178.
Tempel, B.L., Livingstone, M.S., Quinn, W.G., 1984. Mutations in the dopa
decarboxylase gene affect learning in Drosophila. Proc. Natl. Acad. Sci. U. S. A. 81,
3577 – 3581.
Valles, A.M., White, K., 1986. Development of serotonin-containing neurons in
Drosophila mutants unable to synthesize serotonin. J. Neurosci. 6, 1482 – 1491.
Venkatesh, K., Siddhartha, G., Joshi, R., Patel, S., Hasan, G., 2001. Interactions
between the inositol 1,4,5-trisphosphate and cyclic AMP signaling pathways regulate
larval molting in Drosophila. Genetics. 158, 309 – 318.
Walther, D.J., Peter, J.U., Bashammakh, S., Hortnagl, H., Voits, M., Fink, H., Bader,
M., 2003. Synthesis of serotonin by a second tryptophan hydroxylase isoform.
Science. 299, 76 – 76.
Walker, Q.D., Williams, C.N., Jotwani, R.P., Waller, S.T., Francis, R., Kuhn, C.M.,
2006. Sex differences in the neurochemical and functional effects of MDMA in
Sprague-Dawley rats. Psychopharmacol. (Berl). 189, 435 – 445.
213
West, G.B., Woodruff, W.H., Brown, J.H., 2002. Allometric scaling of metabolic rate
from molecules and mitochondria to cells and mammals. Proc. Natl. Acad. Sci. U. S.
A. 99, (Suppl 1), 2473 – 2478.
Whitaker-Azmitia, P. M., Druse, M., Walker, P., Lauder, J. M., 1996. Serotonin as a
developmental signal. Behav. Brain Res. 73, 19 – 29.
White, C.R., Seymour, R.S., 2005. Allometric scaling of mammalian metabolism. J.
Exp. Biol. 208, 1611 – 1619.
Zhang, X., Beaulieu, J.M., Sotnikova, T.D., Gainetdinov, R.R., Caron, M.G., 2004.
Tryptophan hydroxylase-2 controls brain serotonin synthesis. Science. 305, 217.
214
VITA
Ms. Sameera Dasari
EDUCATION: • University of Kentucky 2002-present Doctoral program (Biology)
Specialization: Molecular biology, Neurobiology • Nagarjuna University, India 1999 - 2001
Master of Science Specialization: Biochemistry
• Osmania University, India 1996 – 1999 Bachelor of Science
Specialization: Biochemistry ACADEMIC AWARDS:
• Ribble scholarship (2002-2003) Department of Biology, University of Kentucky, Lexington, KY.
• Junior research fellowship (2001- 2002) The Indian Council of Agricultural Research (ICAR) under the National Professor project, Directorate of Rice Research, Hyderabad, India.
PUBLICATIONS: 1. Dasari, S. and Cooper, R.L. (2004) Modulation of sensory to motor circuits by
serotonin, octopamine, and dopamine in semi-intact Drosophila larva. Neurosci Res. Feb; 48(2): 221-227.
2. Sparks, G.M., Dasari, S. and Cooper, R.L. (2004) Presynaptic actions of MDMA (Ecstasy) at glutamatergic synapses. Neurosci. Res. 48 (4) 431–438.
3. Dasari, S. and Cooper, R.L. (2004) Monitoring Heart Rate in Drosophila Larvae by Various Approaches. Drosophila Information Service. 87:91-96.
4. Dasari, S. and Cooper, R.L. (2005) Direct influence of serotonin on the larval heart of Drosophila melanogaster. J. Comparative Physiology B: Biochemical, Systemic, and Environmental Physiology. 176: 349–357.
5. Dasari, S., Turner, C. and Cooper, R.L. (2005) Influence of the serotonergic system on physiology, development and behavior of Drosophila melanogaster. (In Manuscript).
6. Dasari, S., Cullman-Clark, B., Harrison, F., and Cooper, R.L. (2005) Role of serotonin receptor 5HT2dro on physiology development and behavior of Drosophila melanogaster. (In preparation).
Abstracts:
1. Dasari, S. and Cooper, R.L. (2003) Modulation of sensory to motor circuits by serotonin, octopamine, and dopamine in semi-intact Drosophila larva. Univ. of Kentucky Neurosciences Day, April 28, 2003.
2. Cooper, R.L., Sparks, G.M. and Dasari, S. (2003) CNS and NMJ actions of MDMA (Ecstasy): Cholinergic & Glutamatergic synapses. Society for Neuroscience meeting Nov. 2003.
3. Dasari, S. and Cooper, R.L. (2003) Sensory stimulation in semi-intact Drosophila larva induces CNS activity and recruitment of discernable motor units. Society for Neuroscience meeting, Nov. 2003.
215
4. Dasari, S. and Cooper, R.L. (2004) Effects of serotonin and MDMA (Ecstasy) on neuronal function of Drosophila melanogaster. Univ. of Kentucky Neurosciences Day, March 11, 2004.
5. Dasari, S. and Cooper, R.L. (2004) Effects of serotonin and MDMA (Ecstasy) on neuronal function and heart rate of Drosophila melanogaster. Univ. of Kentucky Naff symposium, Department of chemistry, April 2, 2004.
6. Dasari, S., Cooper, R.L. (2004) Effects of serotonin and MDMA in modulating sensory-CNS-motor circuit in a semi-intact preparation of Drosophila larvae. An international meeting of Drosophila neurobiology. Neuchâtel, Switzerland. Sept 4-8, 2004.
7. Stoeppel, C. O’Connell, S., Hensley, A., Bhatt, D., Logsdon, S., Richardson, G., Johnstone, A., Lancaster, M., Viele, K., Kim, S., Dasari, S.,Cooper, R.L. (2004) Integration of neurophysiology, anatomy and behavior with mathematics & statistics in a workshop course. Society for Neuroscience meeting 2004 San Diego, CA.
8. Dasari, S., Nichols, R., Cooper, R.L. (2004) Effects of 5-HT and MDMA on heart rate of 3 rd instar Drosophila melanogaster. Society for Neuroscience meeting 2004 San Diego, CA.
9. Bhatt, D.M., Dasari, S., Brewer, L.D., Cooper, R.L. (2004) Characterization of Glutamate receptors at the Drosophila neuromuscular junction. Society for neuroscience meeting 2004 San Diego, CA.
10. Dasari, S., Cooper, R.L. (2005) Influence of the serotonergic system on physiology, development, and behavior of Drosophila melanogaster. Society for Neuroscience 2005 meeting Washington, DC.
11. Cullman-Clark, B., Dasari, S., Pauly, J., Cooper, R.L. (2005) Influence of 5-HT receptors on behavior and heart rate in Drosophila melanogaster larvae. Society for Neuroscience 2005 meeting Washington, DC.
12. Turner, C., Dasari, S., and Cooper, R.L. (2005) Influence of the dopamine and serotonergic systems on physiology, development and behavior of Drosophila melanogaster. Kentucky Academy of Sciences. Annual meeting. Eastern KY University, Richmond, KY. Nov. 10-12.
13. Hill, J., Dasari, S., Badre, N., Blackburn, J., Viele, K. 19 Middle School, 2 High School Teachers in Fayette County and Cooper, R.L. (2005) Influence of nicotine on physiology, development and behavior of Drosophila melanogaster: Useful approaches for public school projects. . Kentucky Academy of Sciences. Annual meeting. Eastern KY University, Richmond, KY. Nov. 10-12.
14. Turner, C., Dasari, S., and Cooper, R.L. (2006) Influence of the dopamine and serotonergic systems on physiology, development and behavior of Drosophila melanogaster. Society for Integrative and Comparative Biology annual meeting. Orlando, FL. January 4-8.
15. Cooper, R.L., Dasari, S., Turner, A.C., Cullman-Clark, B., White, J. (2006) Effects of the serotonergic system on physiology, development, learning and behavior of Drosophila melanogaster. VIII East European Conference of the International Society for Invertebrate Neurobiology Simpler Nervous Systems. Kazan, Russia, Sept. 13-17.